Cross-Linkable Glycoproteins and Methods of Making the Same

ABSTRACT

Non-naturally occurring peptides/polypeptides/proteins comprising the crosslinking motif, Tyr-X-Tyr, wherein X is any amino acid, and methods of preparing the same.

This application claims priority to U.S. Provisional Patent ApplicationNos. 60/563,349, filed Apr. 19, 2004, and 60/653,236, filed Feb. 15,2005. The entire disclosures of these applications are incorporatedherein by reference.

FIELD OF THE INVENTION

The invention is directed to methods of making crosslinkable molecules,such as peptides/polypeptides/proteins, by introducing a crosslinkingmotif into the molecule's structure, and the crosslinkable moleculesmade according to these methods.

BACKGROUND OF THE INVENTION The Plant Cell Wall

The plant cell wall is composed of independent, interacting networks ofcellulose microfibrils tethered by hemicellulosic polysaccharides, whichare embedded in a matrix of pectins, glycoproteins, and phenolicsubstances (Carpita and Gibeaut 1-30). The plant cell wall is not animmutable substance; rather, it is a self-(re)organizing barrier able torespond to external and internal stimuli to govern cellular defense,growth, and development. For a size perspective, plant cell walls areapproximately 0.1-10 μm thick depending on the type of plant species. Incomparison, the plasma membranes of plant cells are not more than 0.01μm thick (Stephen C. Fry).

Plant cell walls are generally subdivided into two types: the primarycell wall and the secondary cell wall. Primary cell walls aresynthesized and modified during a period of active cell growth and areoften referred to as “growing cell walls” (Kerr and Bailey 327-49).Secondary cell walls are deposited after cell growth has stopped. Sincecell wall material is deposited from the inside out, the secondary cellwall is interior to the primary cell wall. Although the bulk of matureplant cell walls are composed of secondary wall material, there is greatinterest in the components of the primary cell wall, and theircontrolled assembly and modification, because primary cell walls dictatecell type, shape, and size, and provide a selective barrier to theexternal environment.

The Hydroxyproline-Rich Glycoproteins

The hydroxyproline-rich glycoproteins (HRGPs), which include theextensins, proline-rich proteins, and arabinogalactan-proteins,contribute to the extracellular matrix throughout the plant kingdom andthe Chlorophycean green algae (Kieliszewski and Lamport 157-72;Showalter and Varner 485-520). HRGPs are involved in all aspects ofplant growth and development involving wall architecture (Goodenough etal. 405-17; Roberts 129-46) and wall assembly during embryogenesis (Halland Cannon 1161-72) as well as responses to biotic and abiotic stress(Merkouropoulos and Shirsat 356-66; Merkouropoulos, Barnett, and Shirsat212-19; Yoshiba et al. 115-22) that include mechanical stress (Shirsatet al. 618-24); (Hirsinger et al. 343-55), physical wounding (Chen andVarner 2145-51; Han et al. 59-70; Showalter et al. 547-65; Zhou, Rumeau,and Showalter 5-17), pathogenesis (Benhamou et al. 457-67), andsymbiosis (Cassab 441-46; Franssen et al. 4495-99; Frueauf et al.429-38).

HRGPs are extended macromolecules consisting of small repetitive peptideand glycopeptide motifs that form peptide modules and glycomodules offunctional significance, as in “mix-and-match” mode they define themolecular properties of the overall macromolecule (Kieliszewski et al.538-47; Kieliszewski and Lamport 157-72). The glycomodules result from acombination of posttranslational modifications unique to plants, namelyproline hydroxylation (Lamport 1438-40) and its subsequent glycosylation(Lamport 1322-24) that leads either to short arabinooligosaccharide orlarger arabinogalactan polysaccharide addition to the Hyp residues. Asequence-dependent O-Hyp glycosylation code directs the precise additionof oligosaccharides and polysaccharides (Kieliszewski 319-23) and thereis increasing evidence that other sequence-dependent codes direct inter-and intramolecular crosslinking of HRGPs. Crosslinked HRGPs, includingextensins, contribute to wall architecture and defense responses byforming interpenetrating crosslinked networks in the wall. However, theprecise identity of the intermolecular crosslink(s) has remainedelusive.

The Extensins

Extensins are structural HRGPs that are covalently crosslinked into theprimary cell wall, rendering them insoluble. Aside from being rich inHyp, they also are rich in Ser, Tyr, Lys, Val and Thr. They areextensively post-translationally modified with shortHyp-O-oligoarabinosides and typically possess monogalactosyl-serine(Lamport, Katona, and Roerig 125-31; Lamport and Miller 454-56).Extensins are rigid linear molecules that adopt a polyproline IIleft-handed helical conformation (3 residues per turn, 9.4 Å pitch) (vanHolst and Varner 247-51); (Heckman, Terhune, and Lamport 848-56);(Stafstrom and Staehelin 242-46).

Three major types of extensin precursors to the extensin network arewidespread in dicot plants, namely Precursor 1 (P1) extensins that arecharacterized by the repetitive motif:Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, (Smith et al. 1021-30; Smith,Muldoon, and Lamport 1233-39), P2 extensins that contain repeats of themotif Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys-Tyr-Lys (Smith et al. 1021-30;Smith, Muldoon, and Lamport 1233-39), and finally, the P3 extensins thatcontain a major palindromic (bolded) repeat:Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr-Tyr-Tyr-Lys (Lamport79-115; Smith et al. 1021-30).

The P1 and P2 extensins can be isolated by the salt-elution of intactcells as soluble monomer precursors to the extensin network (Fong et al.548-52; Smith et al. 1021-30; Smith, Muldoon, and Lamport 1233-39). TheP3 extensins, on the other hand, have never been isolated as monomericprecursors to the extensin network, presumably due to their rapidincorporation into the cell wall via covalent intermolecularcrosslinking (Mort and Lamport 289-309). Consequently, the molecularproperties of soluble monomeric P3 extensins have thus far been inferredfrom gene sequences, and P3 glycopeptides and peptides rendered fromcell walls (Corbin, Sauer, and Lamb 4337-44; Lamport 1155-63; Lamport27-31; Lamport 79-115; Lamport and Miller 454-56; Showalter et al.547-65; Showalter and Varner 375-92; Zhou, Rumeau, and Showalter 5-17).

Intramolecular Extensin Crosslinking

Both P2 and P3 type extensins can undergo intramolecular crosslinking ofTyr residues (Epstein and Lamport 1241-46) (underlined in the sequencesabove) to form the diphenylether crosslink amino acid, isodityrosine(IDT) (FIG. 1) (Fry 449-55). First observed as an unknown tyrosinederivative in extensin peptides (Lamport 1155-63), and later identifiedin wall hydrolysates (Fry 449-55), and also as a component oftrityrosine in Ascaris cuticle collagen (Fujimoto 637-43), IDT wasinitially hypothesized to be an intermolecular crosslink responsible fortransforming the soluble extensin monomeric precursors, P1, P2 and P3,into an insoluble extensin network in muro (Fry 449-55); (Lamport andEpstein 73-83). But IDT was identified only as an intramolecularcrosslink in P2 and P3-derived extensin peptides purified from enzymicdigests of cell walls (Epstein and Lamport 1241-46) and extensinpeptides crosslinked by intermolecular IDT have never been isolated.Although it has been suggested that extracellular peroxidases catalyzethe formation of IDT, to date, the precise mechanism in muro remains amystery (Fry 853-62).

Intermolecular Extensin Crosslinking

Evidence has suggested that pectin-protein crosslinks may play a role inextensin insolublization in muro (Keegstra et al. 188-96; Mort; Qi etal. 1691-701). Although these crosslinks may exist to some extent, theextensin network in cell walls and in vitro remains insoluble aftercomplete deglycosylation with anhydrous hydrogen fluoride at 0° C. (Mortand Lamport 289-309; Schnabelrauch et al. 477-89). Furthermore, Hyp-richextensin peptides are only released after proteolytic cleavage of apartially or fully deglycosylated extensin network (Lamport 1155-63;Lamport 79-115; Mort and Lamport 289-309; Qi et al. 1691-701). Thus, theinsolubility of cell wall extensins is primarily attributed to aprotein-protein and/or protein-phenolic-protein crosslink(s) (Mort andLamport 289-309).

Peroxidase-catalyzed extensin intermolecular crosslinking has beendemonstrated in vitro by several groups (Brownleader et al. 1115-23;Everdeen et al. 616-21; Jackson et al. 1065-76; Price et al. 41389-99;Schnabelrauch et al. 477-89), yet the molecular nature of theintermolecular crosslink(s) has not been were not identified. Indeed,Lamport and colleagues (Schnabelrauch et al. 477-89) found no IDTincrease in P1 extensins after their crosslinkage in vitro by a tomatopl 4.6 extensin peroxidase, although the abundance of Val-Tyr-Lys motifsin several crosslinking extensins, including P1 and P2, suggested theintermolecular crosslinks involved tyrosine and/or lysine (Schnabelrauchet al. 477-89).

More recently, Brady and Fry identified a trimeric tyrosine derivative,pulcherosine (FIG. 2) and the tetrameric tyrosine derivative,di-isodityrosine (dilDT; FIG. 3) (Brady, Sadler, and Fry 349-53);(Brady, Sadler, and Fry 323-27) in tomato cell wall hydrolysates andspeculated that IDT-containing extensins could be insolubilized throughintermolecular IDT crosslinks forming pulcherosine and di-isodityrosine(FIG. 4). Also, as extensin incorporation into the cell wall increased,hydrolysates of these walls showed that the amounts of IDT decreased anddilDT increased (Brady and Fry 87-92). These findings suggest asignificant role for IDT-rich extensins, which are supported by therecent discovery that RSH, an extensin containing 14 intramolecular IDTmotifs, is crucially involved in positioning the cell plate during theearliest stages of embryogenesis in Arabidopsis (Hall and Cannon1161-72).

However, to date, there has been no direct demonstration of an extensinintermolecular crosslink that involves either Tyr or Lys. The results ofin vitro assays have been difficult to interpret, as the substrateextensins, P1 and P2, contain both Tyr and Lys and the amino acidsformed by crosslinking were not identified (Brownleader et al. 1115-23;Everdeen et al. 616-21; Fujimoto 637-43; Hall and Cannon 1161-72;Schnabelrauch et al. 477-89). Other approaches involving the isolationof intermolecularly crosslinked peptides from the cell wall itself havealso proven intractable (Epstein and Lamport 1241-46).

SUMMARY OF THE INVENTION

The present invention advances the art by, among other things,identifying a residue involved in glycoprotein crosslinking.

In order to simplify the results of in vitro crosslinking assays and totest for the involvement of Tyr and Lys in crosslinking, we created aseries of extensin mutants using the synthetic gene approach describedearlier (Kieliszewski 319-23; Shpak et al. 11272-78; Shpak et al.11272-78; Shpak, Leykam, and Kieliszewski 14736-41; Tan L 1362-69; Tan,Leykam, and Kieliszewski 1362-69). The P3 extensin was of particularinterest in view of its repetitive structure widespread in the plantkingdom and therefore functionally significant although frustratinglyrecalcitrant to isolation in precursor form; furthermore, it containsTyr, Lys, and an intramolecular IDT motif. The synthetic gene approachallows us to explore the roles of IDT, Tyr, and Lys in crosslinking, andeventually to test the proposed role of the palindromic motif inmolecular recognition (Kieliszewski and Lamport 157-72).

Thus, we designed a series of synthetic genes, one encoding twenty andthe other eight repeats of the P3 repetitive subdomain:Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr-Tyr-Tyr-Lys, as wellas variants containing Tyr→Phe and Lys→Leu mutations. Overexpression ofthese genes as enhanced green fluorescent protein (EGFP) fusion proteinsin BY-2 tobacco cells yielded secreted transgenic extensins. They wereglycosylated as predicted by the Hyp contiguity hypothesis and containedIDT similar to native P3-type extensins. Furthermore, the pl 4.6extensin peroxidase (E.C. 1.11.1.7) (Schnabelrauch et al. 477-89)catalyzed intermolecular crosslinking only between those P3 extensinanalogs which contained Tyr. The present invention advances the art by,among other things, identifying a particular residue, i.e., tyrosine,involved in crosslinking.

Thus, by using consensus sequences that are targeted for crosslinking,novel molecules can be created, which will form crosslinks. Theconsensus sequence for crosslinking is Tyr-X-Tyr, where X is any aminoacid. In some embodiments, X is chosen from Tyr, Lys, and Val. Thecrosslinks that can form with this motif can be intermolecular orintramolecular. By inserting these sequences into proteins,crosslinkable proteins are generated.

The present invention provides, for example, crosslinked fibers,emulsifiers, moleclular scaffolds, etc. Other specific applications ofthe invention include, but are not limited to, biologically activefilms. For example, a crosslinkable chimera involving an enzyme at oneend (e.g., nitric oxide synthase or peroxidase or any other enzyme) andcrosslinking extensin repeats at the other, would allow for animmobilized enzyme in a film to make a biosensor or for implantation ata wound site. Other immobilized proteins include, but are not limitedto, lectins (which bind carbohdrate residues) and protein sequences thatcan chelate metals, etc. It is also contemplated that arabinogalactanmodules (X-Hyp-X-Hyp) with extensin arabinosylation modules (X-Hyp_(n))can be combined with crosslinking modules and with non-HRGP modules tocreate novel combinations.

The present invention provides non-naturally occurring proteinscomprising the amino acid sequence Tyr-X-Tyr, wherein X is chosen fromany amino acid. In some embodiments, X is chosen from Tyr, Lys, and Val.In some embodiments, the protein is intermolecularly crosslinked to anon-naturally occurring protein comprising the amino acid sequenceTyr-X-Tyr. In some embodiments, the non-naturally occurring protein isintramolecularly crosslinked to itself.

The non-naturally occurring proteins of the invention can furthercomprise the amino acid sequence X-Hyp_(n), wherein X is any amino acid,and n is from 1 to about 1000. In some embodiments, X is chosen fromSer, Ala, Val, and Thr. The non-naturally occurring proteins of theinvention can also further comprise the amino acid sequence X-Hyp-X-Hyp,wherein X is any amino acid. In some embodiments, X is chosen from Ser,Ala, Val, and Thr.

The non-naturally occurring proteins of the invention can be crosslinkedby reactions that involve the oxidation of tyrosine. Such reactions canbe catalyzed by peroxidases, including but not limited to, horseradishperoxidase, tomato peroxidase, extensin peroxidase, etc.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) ofthe invention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of isodityrosine (IDT). (MW=360;(Fry, S. C. 1982))

FIG. 2 shows the chemical structure of pulcherosine. (MW=539; (Brady, J.D. et al. 1998))

FIG. 3 shows the chemical structure of diisodityrosine (dilDT). (MW=718;(Brady, J. D. et al. 1996))

FIG. 4 illustrates a possible in muro mechanism of pulcherosine anddilDT formation. From Brady et al. 1996, 1997, and 1998, pulcherosinemust come from the oxidative coupling of 1 IDT with yr; DilDT must comefrom the oxidative coupling of either pulcherosine and Tyr or 2 IDTs, asbiphenyl linked dityrosine (upper right, boxed) is not found in plants.

FIG. 5 shows oligonucleotide sets for construction of the syntheticgenes: (A) FK9, (B) YK, (C) YL, and (D) FL. (A) Construction of the FK9gene involved three sets of partially overlapping, complementaryoligonucleotide pairs that were polymerized head-to-tail as describedearlier (McGrath, K. P. et al. 1990) (Shpak, E. et al. 1999). Nineinternal repeats and one of each linker set made up the FK9 syntheticgene. The restriction sites used for subcloning are highlighted andlabeled. The YK, YL, and FL genes were constructed through primerextension of the sense and antisense oligonucleotide sets shown in (B),(C), and (D) which involved elongation through use of the complementarybut nonregenerable BbsI and BsmFl restriction sites. As the enzymes BbsIand BsmFl do not restrict the site that they recognize, both therecognition sequence and the sequence that is restricted are highlightedwith the same color, the label directly placed above the recognitionsite. The underlined sequences in (B) (C) and (D) indicate the 24 basepair overlapping sections of the oligonucleotide sets.

FIG. 6 illustrates a plasmid map of the pUC18-FK9 plasmid. The FK9synthetic gene was inserted as BamHI-EcoRI fragment into a pUC18 vector.

FIG. 7 illustrates the construction of the YK20, YK8, and YL8 usingoligonucleotides containing complementary, nonregenerable restrictionsites. This approach used general methods developed earlier (Lewis, R.V. et al. 1996). Primer extension of the sense and antisense overlappingoligonucleotide sets shown in FIGS. 5B, C, and D (here designated YK-ssand YK-as, respectively) initially gave genes having a single repeat(upper left) which were inserted into pUC18 as XmaI/SacI fragments andthen sequenced. The resulting plasmid, here designated pUC18-YK, wasdivided into two aliquots; one aliquot was restricted with BbsI and ScaIand the other with BsmFl and ScaI. The BbsI/ScaI and BsmFl/ScaIfragments were isolated, annealed to each other through thecomplementary nonregenerable BbsI/BsmFl site and the ScaI site, thenligated to give rise to pUC18-YK2. Longer P3 analog genes were made byrepeating the procedure.

FIG. 8 illustrates a plasmid map of pUC18-YK. The YK synthetic gene wasinserted as an XmaI-SacI fragment into the pUC18 plasmid. This procedurewas also employed for the synthesis of pUC18-YL and pUC18-FL.

FIG. 9 illustrates a plasmid map of the modified pUC-SStob-YK20-EGFP.Synthetic genes YK20 (shown here), YK8, YL20, YL8, and FL8 were insertedas XmaI-NcoI fragments into the modified pUC-SStob-EGFP vector betweenSStob and the EGFP reporter gene.

FIG. 10 illustrates a plasmid map of recombinant pBI121-SStob-YK20-EGFP.Synthetic genes SStob-YK20-EGFP (shown here), SStob-YK8-EGFP,SStob-YL20-EGFP, SStob-YL8-EGFP, SStob-FL8-EGFP, and SStob-FK9-EGFP wereinserted as BamHI-SacI fragments into the pBI121 binary planttransformation vector replacing the β-glucuronidase gene as described byShpak, E. et al. 1999. Nos-pro: nopaline synthesis promoter; NOS-ter:nopaline synthesis terminator; NPTII: Neomycin Phosotransferase II gene.

FIG. 11 shows oligonucleotide sequences of PCR and DNA sequencingprimers. (a) M13 forward primer is complimentary to 21 bp upstream ofthe MCS region of pUC 18 and was used for sequencing synthetic genes inpUC vectors. (b) The tobacco signal sequence primer (SSeq4Sen) binds 4bp into the signal sequence and was used for PCR of genomic DNA fromtransformed cell lines as well as the DNA sequencing of the PCRproducts. (c) The EGFP primer (EGFP566 as) is complimentary to the sensestrand of the EGFP gene 566 bp from the origin. This oligonucleotide wasused for PCR of genomic DNA and the sequencing of synthetic genes inboth pUC and pBI121 vectors. All oligonucleotide primers were orderedfrom Integrated DNA Technologies.

FIG. 12 is a flowchart for the isolation of EGFP fusion glycoprotein.(diagram courtesy of Dr. Li Tan). Culture medium of transformed tobaccocells was concentrated and processed by HIC, gel filtration and reversedphase HPLC.

FIG. 13 shows the DNA and encoded protein sequences of the P3-typeextensin analogs. (A) SStob-YK8-EGFP and SStob-YK20-EGFP, (B)SStob-YL8-EGFP and SStob-YL20-EGFP, (C) SStob-FK9-EGFP and (D)SStob-FL8-EGFP. Each gene encoded a signal sequence, a P3 extensinanalog, and EGFP, which is presented only in part. The XmaI and NcoIsites allowed insertion of the genes into the modified pUC18 vectordescribed earlier (Shpak, E. et al. 1999) between the signalsequence-encoding region (SStob) and EGFP. The BamHI site together witha SacI site (not shown) located at the 3′ end of EGFP allowed insertionof SStob-YK20-EGFP and the other constructions into the binary planttransformation vector, pBI121.

FIG. 14 shows PCR amplification of genomic DNA from cell linesNtYK20-EGFP and NtYL8-EGFP. Insertion of the synthetic genesSStob-YK20-EGFP and SStob-YL8-EGFP into the tobacco genome was verifiedby PCR amplification with oligonucleotide primers complimentary to theantisense strand of SStob and the sense strand of EGFP. Bands at 783 bpand 1359 bp from lanes 1 and 2 respectively were eluted from the gel andsequenced. Lane 1: SStob-YL8-EGFP PCR product; Lane 2: SStob-YK20-EGFPPCR product; Lane 3: untransformed tobacco PCR product (control); Lane4: 100 bp ladder.

FIG. 15 is a visualization of cell lines (A) NtYK20-EGFP, (B)NtYK8-EGFP, (C) NtYL8-EGFP, (D) NtFK9-EGFP, (E) uTob, and (F) NtSS-EGFPby fluorescence microscopy. Slides shown here are an overlay of thelaser scanned image (exc. 488 nm, emm. 510 nm) and the transmitted lightimage.

FIG. 16 is a visualization of plasmolyzed SS-EGFP, uTob, and YK20-EGFPcells by fluorescence microscopy. The (A) SS-EGFP, (B) uTob, (C and D)YK20-EGFP cells were plasmolyzed with 750 mM mannitol. The (E) YK20-EGFPcells were plasmolyzed with 500 mM potassium phosphate pH 7. Slidesshown here are an overlay of the laser scanned image (exc. 488 nm, emm.510 nm) and the transmitted light image. Green fluorescence in the cellwalls after plasmolysis was not observed for cells expressing YK8-EGFP,YL8-EGFP, and FK9-EGFP.

FIG. 17 shows a fractionation of transgenic NtYK20-EGFP culture mediumby HIC. Concentrated and desalted NtYK20-EGFP (shown here), NtYK8-EGFP,NtYL8-EGFP, and NtFK9-EGFP culture media were loaded onto a HIC columnand eluted stepwise from 2 M NaCl to 1 M NaCl and then to water. Theeluate was monitored by in-line fluorescence detection (exc. 488 nm,emm. 510 nm; Hewlett Packard, USA). A single green peak eluted in 1 MNaCl.

FIG. 18 shows purification of the YK20-EGFP, YK8-EGFP, YL8-EGFP, andFK9-EGFP fusion glycoproteins by C4 reversed phase HPLC. (A) YK20-EGFP(300 μg), (B) YK8-EGFP (200 μg), (C) YL8-EGFP (200 μg), and (D) FK9-EGFP(45 μg) were injected onto an analytical C4 reversed phase HPLC column.The fusion glycoproteins eluted between 28 and 32 min (58% and 68% endbuffer).

FIG. 19 shows the isolation of glycomodules YK20, YK8, YL8, and FK9 byC4 reversed phase HPLC. Glycomodules YK20, YK8, YL8, and FK9 elutedbetween 30-40% end buffer, prior to their respective undigested fusionglycoproteins.

FIG. 20 shows a C4 reversed phase HPLC purification of dYK20-EGFP.Deglycosylated YK20-EGFP (200 μg) was purified on a C4 analyticalcolumn. The dYK20-EGFP fusion protein showed a slightly increasedretention time (˜69% end buffer) compared to the respective fusionglycoprotein.

FIG. 21 demonstrates an examination of acid hydrolysates of (A) YK20,(B) YK8, (C) YL8 glycomodules and (D) FK9-EGFP fusion glycoprotein forIDT. Acid hydrolysates (10 μg each) were fractionated by SEC. The YK20,YK8, and YL8 glycomodules contained IDT whereas FK9-EGFP did not.

FIG. 22 shows the detection of Hyp-PS in base hydrolysates of YL8-EGFPfractionated by Superdex-75 gel filtration chromatography. A HMWHyp-O-glycoside peak was observed in the hydrolysate of YL8-EGFP elutingbetween 18 and 38 min. This confirmed the presence of Hyp-PS attachmentto YL8-EGFP.

FIG. 23 shows the purification of the pl 4.6 extensin peroxidase byanion exchange HPLC. Heme absorbance was monitored at 405 nm, and asingle peak eluted at ˜50% B (250 mM NaCl).

FIG. 24 shows in vitro crosslinking of P1 extensin (40 μg) by the pl 4.6extensin peroxidase. Crosslinking reactions at time zero (A) and after15 minutes of incubation (B) were separated by Superose-6 gel filtrationchromatography. Peaks eluting after 22 min are a result of thecrosslinking buffer and stopping reagent. P1, monomic P1 extensin; P1XL,crosslinked P1 extensin.

FIG. 25 shows in vitro crosslinking of YK20 (40 μg) by the pl 4.6extensin peroxidase. Crosslinking reactions at time zero (A) and after15 minutes of incubation (B) were separated by Superose-6 gel filtrationchromatography. YK20, monomeric YK20; YK20XL, crosslinked YK20. The pl4.6 extensin peroxidase crosslinks YK20 in vitro.

FIG. 26 shows in vitro crosslinking reactions of FK9 (40 μg).Crosslinking reactions at time zero (A) and after 15 minutes ofincubation (B) were separated by Superose-6 gel filtrationchromatography. Note FK9 did not crosslink in vitro. FK9 mono,monomeric.

FIG. 27 shows in vitro crosslinking reactions using BSA (40 μg) as acontrol substrate. Crosslinking reactions were incubated 0 min (A) and15 min (B) then separated by Superose-6 gel filtration chromatography.BSA did not crosslink.

FIG. 28 shows in vitro crosslinking reactions of YK20 (40 μg) lackingthe extensin peroxidase enzyme. Crosslinking reactions lacking theaddition of peroxidase were incubated 0 min (A) and 15 min (B) thenseparated by Superose-6 gel filtration chromatography. YK20 does notcrosslink in this time frame without the addition of extensinperoxidase.

FIG. 29 shows in vitro crosslinking reactions of YK20 (40 μg) lackingthe H₂O₂ co-substrate. Crosslinking reactions lacking the addition ofhydrogen peroxide were incubated 0 min (A) and 15 min (B) then separatedby Superose-6 gel filtration chromatography. YK20 does not crosslinkwith out the addition of H₂O₂.

FIG. 30 shows in vitro crosslinking reactions lacking extensinsubstrate. Crosslinking reactions were incubated 0 min (A) and 15 min(B) then separated by Superose-6 gel filtration chromatography. Notethat peaks eluting after ˜22 min are a result of the buffer and stoppingreagents.

FIG. 31 shows crosslinking rates of P1, YK20, YK8, YL8, and FK9.Crosslinking rates were calculated as described earlier (Everdeen, D. S.et al. 1988) using the first order rate equation A=Ao*e−kt where A isthe monomer remaining (μg) after t (sec) of incubation; Ao is the amountof monomer (μg) at time zero; k is the first order rate constant.

FIG. 32 shows in vitro crosslinking of YK20 by a crude cationic voidperoxidase preparation. Reactions were incubated 0 min (A) and 30 min(B), then separated by Superose-6 gel filtration (0.5 ml/min; OD 220nm). Some crosslinking was observed for YK20 (shown here), P1, and YL8.

FIG. 33 shows size exclusion chromatography of (A) YK20 acid hydrolysateand (B) YK20XL acid hydrolysate. A putative crosslinking amino acid(unknown) eluting at 10.4 min was isolated from the hydrolysate ofYK20XL.

FIG. 34 shows a 1-D ¹HNMR spectrum of the “unknown.” The spectrum wasobtained by Dr. Li Tan of Ohio University. (A) N-linked protonresonances, (B)-(E) aromatic proton resonances.

FIG. 35 shows a predicted 1-D ¹HNMR spectrum of dilDT.

FIG. 36 diagramatically illustrates the pBI121 plasmid with signalsequence-synthetic gene-EGFP. The method of gene construction wasadapted from Shpak et al. (Proc. Natl. Acad. Sci., 1999, 96,14736-14741) Overlapping oligonucleotide pairs were annealed andpolymerized. The synthetic gene was inserted into pUC18 as a BamHI-EcoRIfragment, sequenced and then inserted between the signal sequence andEGFP in pUC-SS-EGFP as a XmaI-NcoI fragment. Finally, the signalsequence-synthetic gene-EGFP unit was placed in the plant transformationvector pBI121 as a BamHI-SacI fragment.

FIG. 37 is a photomicrograph of cell lines expressing EGFP fusionproteins. Panels A. [VYK]₆ and B. [VFL]₆ are transformed cell lines thatshow EGFP fluorescence when viewed with a confocal laser scanningfluorescence mircroscope, excitation 488 nm and emission 510 mn.

FIG. 38 shows results of cross-link assays on a Superose 6 gelfiltration column. Superose 6 profiles of P1 extensin at 220 nm: (a)before and (b) after cross-linking for 15 minutes. Superose 6 profilesof VYK module at 220 nm: (c) before and (d) after cross-linking for 19hours. As cross-linking occurs, the monomer peak decreases and a peakrepresenting a larger cross-linked oligomer appears.

FIG. 39 is a flowchart of oligonucleotide construction.

FIG. 40 shows size exclusion chromatography before (top frame) and after(bottom frame) 30 minutes of crosslinking of (AP)₄(YK)₂₀ catalyzed byextensin peroxidase.

FIG. 41 shows chromatographs of the cross-linking reaction of(AP)₄(YK)₂₀EGFP.

FIG. 42 shows a flow chart for gene construction.

DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to the present embodiments(exemplary embodiments) of the invention.

The following abbreviations are used throughout this application: 1-D¹HNMR: 1-D proton nuclear magnetic resonance; ABTS:2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); ACS: Americanchemical society; BAW: Butanol:acetic acid:water (12:3:5) by volume; bp:base pair; BSA: Bovine serum albumin; BY-2: Bright yellow-2 Nicotianatabacum; CaMV 35S: Cauliflower mosaic virus 35S promoter; CHO:carbohydrate; Da: Daltons; ddH₂O: distilled deionized water; DEAE:diethylaminoethyl; dilDT: diisodityrosine; E. Coli: Escherichia coli;E1: crude peroxidase preparation from untransformed tobacco culturemedium; E2: crude peroxidase preparation from salt-eluates ofuntransformed tobacco cells; % GC: percentage of guanine and cytosinebase pairs; EGFP: enhanced green fluorescent protein; emm: emissionwavelength; exc: excitation wavelength; Fry's dilDT: authenticdiisodityrosine standard from Dr. Stephen C. Fry; HF: anhydrous hydrogenfluoride; HIC: Hydrophobic interaction chromatography; HMW: Highmolecular weight; HPLC: High performance liquid chromatography; HRGP:Hydroxyproline-rich glycoprotein; Hyp: Hydroxyproline; Hyp-O-Aran:O-linked hydroxyproline oligoarabinosides; Hyp-PS: O-linkedhydroxyproline arabinogalactan polysaccharide; IDT: isodityrosine; LB:Luria broth; MALDI-TOF MS: Matrix assisted laser desorptionionization-time of flight mass spectrometry; MCS: Multiple cloningsequence; MS: Mass spectrometry; MW: Molecular weight; MWCO: Molecularweight cut-off; My dilDT: Diisodityrosine standard isolated by theinventor; NG-Hyp: Non-glycosylated Hydroxyproline; NOS pro: nopalinesynthesis promoter; NOS ter: nopaline synthesis terminator; NPT II:Neomycin phosphotransferase II gene; Nt: Nicotiana tabacum; NT-1:Nicotiana tabacum 1 culture medium; P1: Precursor 1 extensin; P1XL:crosslinked P1 extensin; P2: Precursor 2 extensin; P3: Precursor 3extensin; PCR: Polymerase chain reaction; PCV: Packed cell volume; PITC:phenylisothiocyanate; PTC-aa: phenylthiocarbamyl-amino acid derivative;Rf: relative to the solvent front; RSH: Arabidopsis mutant“Root-Shoot-Hypocotyl” defective; SEC: Size exclusion chromatography;Ser-O-gal: O-linked monogalactosyl-serine; SH: Schenk and Hildebrandt;SH*: SH medium lacking kinentin; SStob: tobacco signal sequence; TaOpt:Optimum annealing temperature; TFA: Trifluoroacetic acid; Tm: Meltingtemperature; uTob: untransformed tobacco BY-2 cells; YK20: YK20glycomodules; and YK20XL: crosslinked YK20 glycomodule.

Earlier work using a synthetic gene approach allowed us to elucidate thecodes that drive Hyp-O-glycosylation in plants (Shpak et al. 11272-78;Shpak, Leykam, and Kieliszewski 14736-41; Tan, Leykam, and Kieliszewski1362-69). Here we have extended this work to the extensins, not only asanother test of the Hyp contiguity hypothesis for glycosylation, butalso to examine codes for other posttranslational modifications,including intra- and intermolecular extensin crosslinking, which is along-standing and formidable problem relevant to plant growth,development, and disease resistance. Thus, we designed a series ofcrosslinking and non-crosslinking P3-type extensin analogs for theirsubsequent expression and isolation.

P3-type extensins play a crucial role in the cell wall, judging fromtheir ubiquity and their highly conserved repetitive sequences thatinclude palindromic (bolded) and IDT motifs (underlined):Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr-Tyr-Tyr-Lys. Suchsupersymmetry combined with potential intra- and intermolecularcrosslink sites involving IDT may enhance self-assembly of tightlypacked networks (Knupp and Squire 558-77). Likewise, the related RSHextensin required for cell plate orientation during cytokinesis (Halland Cannon 1161-72) contains 14 IDT motifs and a repetitive symmetrythat, although not palindromic, should favor close packing.

Two methods were used to make the synthetic genes. One involvedpolymerization of duplex DNA oligomer sets (the FK set in FIG. 1A)(McGrath et al. 186-92; Shpak, Leykam, and Kieliszewksi 14736-41), theresult being small genes containing at most only nine internal repeats.A second approach (Lewis et al. 400-06) was used to make the YK20, YK8,and YL8 genes and allowed precise control of the sizes. This strategyinvolved compatible but nonregenerable restriction sites to generate agene with double the number of repeats of the starting gene (FIG. 7).Judging from extensin genes, it was estimated that 20 repeatsrepresented a ‘full length’ extensin, containing about 320 amino acids(Zhou, Rumeau, and Showalter 5-17), although there is considerablevariation in extensin lengths. Therefore, one construction, YK20, wasmade containing 20 palindromic P3 crosslinking modules. Shortervariants, namely YK8, YL8 and FK9, were also made to determine if thenumber of repeats influenced crosslinking rates and to determine theamino acid requirements for crosslinking. This method is completelyamenable to any insert sequence (HRGP or other), for example, by usingBbsI and BsmFl restriction enzymes that have separate recognition andrestriction sites. This method also allows the design of synthetic geneshaving more than one consensus sequence. For instance, repeat units canbe combined to control crosslink densities and/or to create modules suchas AGP/extensin chimeras.

Transformation of tobacco BY2 cells with the synthetic genes yieldedseveral cell lines for each construction. Cell lines releasing the mostfusion protein into the medium were chosen for isolation and biochemicalcharacterization of the transgene products. The yields were on the levelof those previously reported (Shpak et al. 11272-78; Tan L 1362-69; Zhaoet al. 431-44). Transformed tobacco cells were maintained in SH mediumlacking kinetin as this seemed to improve secretion and overall fusionprotein yields as compared with SH medium (not empirically determined).

A combination of HIC, gel filtration, and reversed phasechromatographies (FIG. 18) gave pure fusion proteins judging byN-terminal sequence analyses that yielded a single sequence for eachprotein (Table 5). The amino acid compositions of the isolated YK20,YK8, YL8, and FK9 glycoproteins were also consistent with thosepredicted by the genes (Table 4) although values for Hyp/Pro and Serwere somewhat lower than predicted. Virtually every Pro residue washydroxylated and the modules containing Tyr showed conversion of Tyr toIDT. The PTC-isodityrosine was extremely hydrophobic and eluted with andwas indistinguishable from the reagent peaks at the end of the gradientduring amino acid analysis. Therefore, IDT was quantified from acidhydrolysates fractionated on a gel filtration column (Schnabelrauch etal. 477-89) (FIG. 21) compared to an authentic IDT standard (a gift fromDr. Derek Lamport). Putative IDT motifs of extensins are widespread andquite variable throughout plant species (Table 15). Comparison of theamounts of IDT in YK8 and YL8, show that not all -Y-Y-Y-X- motifs formIDT to the same extent. In other words, the flanking X residue (K or L)appears to influence IDT formation. These crosslinking motif subtletiesmay represent another level of control over wall crosslinking andassembly.

TABLE 15 Putative IDT motifs found in extensin genes of various plants.Putative IDT Motif Plant species YYYK Brassica napus, Phaseolusvulgaris, Solanum tuberosum, Vigna unguiculata, Glycine max, Manihotesculenta, Lycospersicon esculenta, Cicer arietinum, Bromheadiafinlaysonia, Petroselinum crispum, Medicago trucatula, Pisum sativum,Vicia faba YYYH Brassica napus, Phaseolus vulgaris, Glycine max, Manihotesculenta, Lycospersicon esculenta, Cicer arietinum, Arabidopsisthaliana YYYS Brassica napus, Lycospersicon esculenta, Petroselinumcrispum, Arabidopsis thaliana, Nicotiana sylvestris, Oryza saliva YYYQBrassica napus, Phaseolus vulgaris, Petroselinum crispum, Pisum sativum,Lupinus angustifolius YYYN Brassica napus, Phaseolus vulgaris, Vignaunguiculata, Arabidopsis thaliana YYYY Phaseolus vulgaris, Oryza sativa,Lupinus angustifolius YYYT Solanum tuberosum, Petroselinum crispum, YYYVPhaseolus vulgaris YYYI Bromheadia finlaysonia YYYL Arabidopsis thalianaYHY Brassica napus, Phaseolus vulgaris, Glycine max, Lycospersiconesculenta, Cicer arientinum, Arabidopsis thaliana, Pisum sativum,Nicotiana tabacum YHYV Phaseolus vulgaris ,Glycine max, Cicer arietinum,YHYS Pisum sativum, Catharanthus roseus YHYT Glycine max, Cicerarietinum YHYY Catharanthus roseus, Nicotiana sylvestris, YHYQ Cicerarietinum YHYH Arabidopsis thaliana YHYE Daucus carrota YVYK Brassicanapus, Solanum tuberosum, Vigna unguiculata, Glycine max, Lycospersiconesculenta, Arabidopsis thaliana, Lupinu angustifolius, Adiantumcapillus-veneris, Catharanthus roseus YVYS Vigna unguiculata,Arabidopsis thaliana, Pisum sativum, Nicotiana sylvestris YVYQ Brassicanapus YVYG Vigna unguiculata YVY Arabidopsis thaliana YVYN Arabidopsisthaliana YVYH Arabidopsis thaliana YVYA Nicotiana tabacum YKYK Phaseolusvulgaris, Solanum tuberosum, Vigna unguiculata, Glycine max, Pisumsativum, Adiantum capillus-veneris, Catharanthus roseus, Nicotianasylvestris, Daucus carota YKYS Phaseolus vulgaris, Pisum sativum,Catharanthus roseus YKYP Phaseolus vulgaris, Glycine max, Pisum sativum,Vicia faba YKYN Phaseolus vulgaris, Pisum sativum YKYY Lycospersiconesculenta YKYQ Pisum sativum YIYK Phaseolus vulgaris, igna unguiculata,Glycine max, Cicer arietinum, Arabidopsis thaliana, Lupinusangustifolius, Adiantum capillus-veneris, Catharanthus roseus YIYAPhaseolus vulgaris, Glycine max, Manihot esculenta, Cicer arietinum,Arabidopsis thaliana, Pisum sativum YIYS Phaseolus vulgaris, Glycinemax, Bromheadia finlaysonia, Arabidopsis thaliana YIYG Lycospersiconesculenta YIYN Arabidopsis thaliana YLYK Arabidopsis thaliana, Lupinusangustifolius, Adiantum capillus-veneris, Nicotiana tabacum YLYS Cicerarietinum, Arabidopsis thaliana, Pisum sativum, Lupinus angustifoliusYLYN Vigna unguiculata YLYT Lycospersicon esculenta YLYA Nicotianatabacum YSYS Phaseoulus vulgaris, Vicia faba YSYA Phaseoulus vulgarisYSYD Lycospersicon esculenta YSYN Oryza sativa YSYT Daucus carota YPYLVigna unguiculata YPYT Arabidopsis thaliana YPYS Arabidopsis thalianaYDYT Lycospersicon esculenta YDYN Arabidopsis thaliana YEYK Brassicanapus, Vigna unguiculata, Arabidopsis thaliana YEYS Arabidopsisthaliana, Oryza sativa YGYT Zea diploperennis YGYG Zea mays YQYK Vignaunguiculata, Adiantum capillus-veneris YQYS Nicotiana tabacum YAYKLupinus angustifolius YFYS Brassica napus, Adiantum capillus-venerisYMYK Brassica napus YNYS Arabidopsis thaliana YTYS Lycospersiconesculenta, Daucus carota A short-nearly exact protein blast ofhttp://www.ncbi.nlm.nih.gov/BLAST/ with the query “SPPPPYYYK” revealedextensin genes having putative IDT motifs in at least 23 different plantspecies.

Because endogenous P3 extensins are normally insoluble in muro, theirprecursor forms have not been isolated. Thus, P3 glycosylation profilescan only be inferred from P3 extensin genes (Zhou, Rumeau, and Showalter5-17) and from Hyp-glycoside profiles of P3 glycopeptides enzymicallyreleased from the walls (Lamport 79-115). The Hyp-contiguity hypothesis(Kieliszewski et al. 2541-49; Kieliszewski and Lamport 157-72; Shpak etal. 11272-78; Shpak, Leykam, and Kieliszewksi 14736-41) predicts that P3extensins, with their abundant contiguous Hyp and nonclustered singleHyp residues, should be extensively arabinosylated mainly with tetra-and triarabinooligosaccharides but lack arabinogalactan polysaccharides(Tables 2 and 3). The monsaccharide compositions and Hyp-glycosideprofiles of YK20-EGFP, YK8-EGFP, YL8-EGFP, and FK9-EGFP were consistentwith earlier P3 glycopeptide profiles (Lamport 1155-63) and with thepredictions for Hyp-glycosylation, the exception being a small amount ofHyp-polysaccharide in YL8-EGFP (Table 3). Judging by earlierHyp-glycosylation profiles of HRGP analogs having only tandem repeats ofthe pentapeptide (Ser-Hyp-Hyp-Hyp-Hyp)_(n) or the dipeptide(Ser-Hyp)_(n) (Shpak et al. 11272-78; Shpak, Leykam, and Kieliszewksi14736-41), it was inferred that the arabinogalactan polysaccharideprobably occurs on 2 out of the 8 lone Hyp residues occupying the center(underlined) of the palindromic repeats:Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr-Tyr-Tyr-Leu.

As essentially all of the Pro residues in the isolated modules werehydroxylated (Table 4), it was determined that YK20 contained about 180Hyp residues, YK8 and YL8 contained about 72, and FK9 about 81. Of thetotal Hyp residues in each glycoprotein, only 3-8 percent (i.e. 3 to 7Hyp residues/glycoprotein) were not glycosylated, which indicated thatoften all Hyp residues in the 16-residue repeats were arabinosylated,including the non-contiguous Hyp. This is consistent with earlier workwhich demonstrated that although contiguous Hyp residues are preferredarabinosylation sites, non-clustered, lone Hyp residues can bearabinosylated or remain non-glycosylated (Kieliszewski et al. 2541-49).

In addition to arabinosides, extensins generally containmonogalactosylated Ser (Lamport 1155-63; Lamport, Katona, and Roerig125-31; Smith, Muldoon, and Lamport 1233-39) and this seems to be truefor YK20, YK8, FK9, and probably for YL8, although at least some of theGal in YL8 occurs in the occasional arabinogalactan adduct present. Themonosaccharide composition of YK20, YK8, and FK9 shows they containedabout 1 mole of Gal for every 10-11 moles of Ara, which suggestedmonogalactose occurred on about 72-90% of the Ser residues in theP3-type module (Tables 7 and 9). This also is in agreement with nativeP3 glycopeptides isolated and characterized earlier from tomato cellwalls (Lamport 27-31).

In Vitro Crosslinking of the P3 Extensin Glycomodules

It was discovered that YK20 was an excellent crosslinking substrate forthe well-characterized tomato pl 4.6 extensin peroxidase (Schnabelrauchet al. 477-89), with rates only somewhat lower than those for tomatoextensin P1, which is presumably a natural substrate of the enzyme(FIGS. 25 and 31). Earlier it was proposed that valine, as in theputative P1 crosslinking motif Val-Tyr-Lys, was a requirement forintermolecular crosslinking of extensins by the pl 4.6 extensinperoxidase (Kieliszewski and Lamport 157-72). However, YK20 contained novaline.

Without wishing to be bound by any particular theory, the underlyingprinciple driving both P3 and P1 self-assembly/molecular recognition forsubsequent crosslinking may involve the inherent hydrophobicity of theputative crosslinking sites (Brady, Sadler, and Fry 323-27; Whitesides,Mathias, and Seto 1312-19), Val-Tyr-Lys in P1 and Tyr-Tyr-Tyr in the P3motif. Polymers containing separate hydrophilic and hydrophobic regions,a characteristic of the extensins, self-assemble in aqueous solutionlargely due to the hydrophobic effect which drives non-polar regionsaway from water and toward each other (Whitesides, Mathias, and Seto1312-19). Thus, it is believed that P1, YK20, YK8, and YL8 crosslinkingis facilitated by the alignment of the hydrophobic crosslinking motifswith one another, which might be further favored by the concurrentalignment of the highly regular and hydrophilic Ser-Hyp₄ glycomodules(Brady, Sadler, and Fry 323-27; Kieliszewski and Lamport 157-72). Therapid rate of P3 crosslinking may partially explain why no one hasisolated an endogenous P3-type extensin monomeric precursor to the wallnetwork: it is very rapidly insolubilized in muro. Consistent with thislikelihood was the incorporation of YK20-EGFP into the tobacco cellwall, presumably through crosslinking as the walls retained greenfluorescence after plasmolysis in mannitol or in salt at concentrationsthat normally remove non-covelently bound material from the wall (FIG.16). Since both YK8-EGFP and YL8-EGFP are able to undergo in vitrocrosslinking, it had been expected to see them in the wall as well. Thiscould be due to their size, which has an impact on crosslinking rates(FIG. 31). It is possible that such a small amount of YK8-EGFP andYL8-EGFP is actually crosslinked into the wall that visualization wasnot possible by fluorescence microscopy, as the relatively low pH of thecell wall could have significantly decreased EGFP fluorescence(CLONTECH).

Two other parameters appeared to influence the crosslinking rates of theP3 analogs. The first is molecular length of the substrate. The rates ofYK20 crosslinking, with 320 amino acids per molecule, were much greaterthan those of YK8, which possessed only 128 amino acids/moleculealthough both had the same number of potential crosslink sites permilligram (˜0.17 mole/mg) of material and similar glycosylation profiles(Tables 2 and 3). Secondly, the presence of lysine in theTyr-Tyr-Tyr-Lys motif favored crosslinking judging by the rates for YK8,which were double those of YL8. It is possible that the lysine residuefacilitates enzyme recognition of the crosslink site or assistscatalysis.

Identification of the P3 Intermolecular Crosslink

The question of the crosslink identity arises and the enzyme or enzymesinvolved. Given the number of peroxidases in the extracellular matrix,including those that crosslink extensin in vitro (Jackson et al.1065-76); (Price et al. 41389-99), it is likely that multiple extensinperoxidases exist having different affinities for different types ofextensins and catalyzing a range of crosslinks. This might explain whyperoxidases from tomato crosslink extensins at different rates: thebasic peroxidase characterized earlier by Everdeen and colleagues(Everdeen et al. 616-21)) has a specific activity that is 2 to 3 ordersof magnitude lower for P1 than the acidic pl 4.6 extensin peroxidasecharacterized later (FIG. 32) (Schnabelrauch et al. 477-89). All of theP3 analogs that contained tyrosine could be crosslinked by extensinperoxidase while FK9, which lacked tyrosine but had lysine, did notcrosslink at all (FIGS. 26 and 31). Thus, in the modules tested here,Tyr, but not Lys, is directly involved in crosslinking. These resultsalso indicated that the Val-Tyr-Lys motif is not required for allextensin crosslinking by the pl 4.6 extensin peroxidase (Schnabelrauchet al. 477-89) and suggest that different intramolecular crosslinksoccur in extensin as the motif present in P1 (putatively Val-Tyr-Lys)differs substantially from the P3 motif (Tyr-Tyr-Tyr).

The exclusive involvement of Tyr in crosslinking of the P3 analogsdescribed here raises questions about the involvement of the crosslinkamino acid diisodityrosine (Brady, Sadler, and Fry 323-27).Characterized hydrolysates of crosslinked YK20 identifieddiisodityrosine as the crosslink amino acid produced during crosslinking(FIG. 33), judging by its atomic mass determined by MS (FIG. 34) andother properties (Brady and Fry 87-92) including NMR spectra of theisolated amino acid (Table 14 and FIGS. 34 and 35). Thus, these resultslend strong support to the suggestion of Brady et al. (Brady, Sadler,and Fry 323-27) that diisodityrosine crosslinks are a mechanism forextensin insolubilization in muro.

Finally, this work demonstrates the general utility of a synthetic geneapproach for elucidating the codes that direct the post-translationalmodifications of HRGPs, including glycosylation (Shpak et al. 11272-78;Shpak, Leykam, and Kieliszewksi 14736-41; Tan, Leykam, and Kieliszewski1362-69) and crosslinking. The ramifications range beyond the roles ofextensin in the wall to the module-by-module design of newglycoprotein-based biopolymers that combine the special propertiesconferred by glycosylation and crosslinking.

EXAMPLES Example 1 Technical Basis for Crosslinking Materials andMethods Construction of P3-Type Synthetic Genes Oligonucleotide Design

The oligonucleotides coding for synthetic P3 extensin genes (FIG. 5)were designed taking into consideration known codon biases of nativetobacco extensins (Showalter and Rumeau 247-81). Appropriate restrictionsites for cloning and/or polymerization were designed into theoligonucleotides as described earlier (Lewis et al. 400-06; Shpak,Leykam, and Kieliszewski 14736-41). The oligonucleotide sequences wereanalyzed for secondary structure formation (i.e. hairpin and dimerformation), annealing temperature optimum (TaOpt), melting temperature(Tm), and false priming sites using Primer Premier software (BiosoftInternational, Palo Alto, Calif.) (Shpak, Leykam, and Kieliszewski14736-41). The oligonucleotide sets were ordered from Integrated DNATechnologies (IDT, Coraliville, Iowa).

Construction of the FK9 Synthetic Gene Annealing of the FKOligonucleotide Pair

Oligonucleotides for the sense and antisense FK internal repeats (FK-sand FK-as respectively; FIG. 5 a) were each dissolved to 0.5 mM insterile distilled, deionized water (ddH₂O). The FK-s and FK-asoligonucleotides (1.5 nmol each) were combined in 30 μl (total volume)of 1×T4 DNA ligase buffer (Promega, Madison, Wis.) and placed in aheating block at 97° C. for 5 min, slowly cooled to 60-65° C. over aperiod of 30 min, then cooled further to 55-60° C. over 30 min. Theheating block was stabilized at the optimum annealing temperature (58°C., calculated using Primer Premier software) for 1 h. The annealed FKinternal repeat pairs were stored at −20° C.

Annealing of the 5′ Linker Oligonucleotide Pairs

The 5′ linker pair contained unique restriction sites for subcloning andwas prepared by the annealing of oligonucleotides (5′-SP3-s) and(5-SP3-as) as previously described (Shpak, Leykam, and Kieliszewski14736-41). Briefly, 4 nmol each of (5′-SP3-s) and (5′-SP3-as) werecombined in 1×T4 DNA ligase buffer to a final volume of 80 μl. Thereaction was heated to 95° C. for 5 min, cooled over 1.15 h to 47° C.,then held at 47° C. for 1.3 h. The annealed 5′ linker pairs were cooledto room temperature then stored at −20° C.

Ligation of the 5′ Linker to the Annealed FK Internal Repeat Pair

The annealed FK internal repeat pairs (0.5 nmol) were combined with theannealed 5′ linker pairs (0.25 nmol) in 1×T4 DNA ligase buffer in asterile screw cap microtube. The sample was heated to 68° C. for 25 min,then 45° C. for 5 min. After cooling to room temperature, 3 U of T4 DNAligase (Promega) was added to the sample (40 μl final vol.). Thereaction was incubated at ambient temperature for 3.6 h, and then cooledto 4° C. for 4.2 h. An additional 1.5 μl (0.075 nmol) of the annealed 5′linker pairs was added and the reaction mixture was incubated further at4° C. overnight (˜14 h). The extent of polymerization was assayed byagarose gel electrophoresis. Excess 5′ linker pairs were removed bySephacryl S-400 microspin column purification according to themanufacturer's instructions (Amersham Pharmacia Biotech, Piscataway,N.J.). Column eluates yielded 5′ linker-FK internal repeats.

To maximize the number of contiguous FK internal repeat pairs ligated toeach 5′ linker pair, an additional 0.4 nmol of FK internal repeat pairswas added to the purified 5′ linker-FK internal repeats along withligase buffer. To re-initiate elongation, the reaction was heated tojust under the Tm (70° C.) for 10 min, followed by 60° C. for 10 min,then 50° C. for 3 h, and finally cooled to room temperature. DNA Ligase(3U, Promega #M1801) was added in a final volume of 50 μl. The reactionwas incubated 30 min at ambient temperature then held at 4° C.overnight.

Ligation of the Annealed 3′ Linker Pair to 5′ Linker-FK Internal Repeats

The annealed 3′ linker pair (FIG. 5 a) was prepared as described for the5′ linker pair. The 5′ linker-FK internal repeats (1.17 nmol) was heatedto 75° C. for 10 min. The annealed 3′ linker pair (0.35 nmol) was addedto the tube while still at 75° C. then returned to the heat block for anadditional 10 min. The sample was slowly cooled, then held at 50° C. for3 h. DNA ligase (3 U) was added at room temperature where it wasincubated 3 h before being transferred to the refrigerator (4° C.).Unincorporated 3′ linkerpairs were removed by Sephacryl S-400 microspincolumns (Amersham Pharmacia). The purified 5′ linker-FK internalrepeats-3′ linker synthetic gene was eluted from the column and storedat −20° C.

Creation of Plasmid pUC18-FK9

The 5′ linker-FK internal repeats-3′ linker synthetic gene was subclonedinto a pUC18 plasmid vector as a BamHI-EcoRI fragment (FIG. 6) (Shpak,Leykam, and Kieliszewski 14736-41). Plasmids were transformed intocompetent E. coli (XL1-Blue, Stratagene La Jolla, Calif.) and selectedfor ampicillin resistance. Positive transformants were cultured in 3.5ml LB medium liquid cultures supplemented 50 μg/ml with ampicillin (220rpm, 37° C.), from which pUC18-FK internal repeats plasmids wereisolated using the Wizard Plus Minipreps, DNA Purification System(Promega, Madison, Wis.). Plasmids were screened for insert size byXmaI-NcoI digestion followed by 1% (w/v) agarose gel electrophoresis. Aplasmid having nine contiguous FK internal repeats was selected (FK9),and sequenced.

Construction of YK, YL, and FL Synthetic Genes

A second method was used to build synthetic genes more similar in sizeto native extensins. This method was adapted from an existing method(Lewis et al. 400-06) and was used to build YK, YL, and FL geneconstructs from new sets of oligonucleotide pairs (FIGS. 5B, C, and D).Restriction sites for BbsI, BsmFl, XmaI, NcoI, and SacI were engineeredat the 5′ and 3′ ends of these new oligonucleotides for subcloning andpolymerization (FIGS. 5B, C, and D).

Dried oligonucleotides were dissolved in ddH₂O to a concentration of 1mg/ml. Corresponding sense (ss) and antisense (as) oligonucleotide pairs(34 μmol each) were combined, and diluted to 20 μl with ddH₂O.Oligonucleotide pairs were heat denatured at 94° C. for 7 min, thenannealed to their respective complimentary 24 base pair annealing sites(60° C. for 5 min, followed by 45° C. for 10 min; FIGS. 5B, C, and Dunderlined). Samples were cooled to room temperature and primerextension was performed using DNA Polymerase I (Klenow) large fragment(Promega) according to manufacturers instructions (room temperature for15 min, then heat stopped at 75° C. for 10 min). Reactions were loadedonto Sephacryl S-200 microspin purification columns (Amersham PharmaciaBiotech) to change the buffer and remove unincorporated nucleotides.Column eluates yielded duplex DNAs designated YK, YL, and FL (FIG. 7).

Creation of Plasmids pUC18-YK, pUC18-YL, and pUC18-FL

The YK, YL, and FL duplex DNAs were each digested with XmaI-SacI,followed by Sephacyl S-400 microspin column purification (AmershamPharmacia Biotech) to remove the small restriction fragment ends(retained on the columns) from duplex DNA monomers. The YK, YL, and FLXmaI-SacI fragments were subcloned to pUC18 to yield plasmids pUC18-YK,pUC18-YL, and pUC18-FL (FIG. 8). Plasmids were sequenced with an M13forward primer.

Creation of Plasmids pUC18-YK20, pUC18-YK8, pUC18-YL20, pUC18-YL8, andpUC18-FL8

Non-regenerable, complimentary sticky ends were produced by separateBsmFl-ScaI and BbsI-ScaI restrictions (New England Biolabs, Beverly,Mass.) of plasmid preparations harboring one repeat of the syntheticgenes (FIG. 7). Ligation of the insert containing BsmFl-ScaI fragmentwith the insert containing BbsI-ScaI fragment produced plasmidspossessing 2 repeats designated pUC18-YK2 and pUC18-YL2 respectively.This process was repeated to achieve plasmids of 8 and 20 internalrepeats (pUC18-YK20, pUC18-YK8, and pUC18-YL8).

Creation of Plasmids pUC18-SStob-YK20-EGFP, pUC18-SStob-YK8-EGFP,pUC18-SStob-YL20-EGFP, pUC18-SStob-YL8-EGFP, pUC18-SStob-FL8-EGFP andpUC18-SStob-FK9-EGFP

Synthetic genes YK20, YK8, YL20, YL8, FL8 and FK9 were inserted betweena tobacco extensin signal sequence (SStob) and the enhanced greenfluorescent protein gene (EGFP) as XmaI-NcoI fragments in the modifiedpUC plasmid (FIG. 9; modified pUC plasmid courtesy of Dr. Elena Shpak)(De Loose et al. 95-100; Shpak, Leykam, and Kieliszewski 14736-41; Tan L1362-69). Plasmids modified pUC-SStob-YK20-EGFP, modifiedpUC-SStob-YK8-EGFP, modified pUC-SStob-YL20-EGFP, modifiedpUC-SStob-YL8-EGFP, modified pUC-SStob-FL8-EGFP, and modifiedpUC-SStob-FK9-EGFP were thus created. These plasmids were againsequenced from the 5′ end using the M13 forward primer and from the 3′end using the 566asEGFP primer (Integrated DNA Technologies). Creationof plasmids pBI121-SStob-YK20-EGFP, pBI121-SStob-YK8-EGFP,pBI121-SStob-YL20-EGFP, pBI121-SStob-YL8-EGFP, pBI121-SStob-FL8-EGFP andpBI121-SStob-FK9-EGFP

The synthetic genes were subcloned into the binary plant transformationvector pBI121 as BamHI-SacI fragments replacing the β-glucuronidasereporter gene to form plasmids pBI121-SStob-YK20-EGFP,pBI121-SStob-YK8-EGFP, pBI121-SStob-YL20-EGFP, pBI121-SStob-YL8-EGFP,pBI121-SStob-FL8-EGFP, and pBI121-SStob-FK9-EGFP (FIG. 10) which werethen transformed to competent E. coli. Positive transformants wereselected on solid LB plates supplemented with kanamycin (30 μg/ml).Plasmids were prepared from 3.5 ml liquid LB culture supplemented with30 μg/ml kanamycin. The control plasmid pBI21-SStob-EGFP, created by Dr.Elena Shpak, was also freshly prepared. Plasmids were screened toconfirm insert size by both BamHI-SacI digestion and XmaI-NcoIdigestions followed by agarose gel electrophoresis (0.7% w/v). Plasmidsthat yielded bands of predicted size in both digestions were selectedfor transformation to Agrobacterium tumefaciens.

DNA Sequencing of Synthetic Genes DNA Sequencing of Synthetic GenesUsing the M13 Forward Primer

We verified insert sequences of pUC18-YK, pUC18-YL, pUC18-FL, pUC18-FK9,modified pUC-SStob-YK20-EGFP, modified pUC-SStob-YK8-EGFP, modifiedpUC-SStob-YL20-EGFP, modified pUC-SStob-YL8-EGFP, modifiedpUC-SStob-FL8-EGFP and modified pUC-SStob-FK9-EGFP by Big Dye TerminatorDNA sequencing (Applied Biosystems, Foster City, Calif.) using the M13forward primer (FIG. 11). Cycle sequencing reactions were preparedaccording to manufacturer's instructions and cycled using either aStratagene-Robocyler Gradient 40-Thermal Cycler or an Applied BiosystemsGeneAmp PCR system 2400. The tubes were placed in the thermocycler andincubated as in Table 1.

TABLE 1 Thermocycler program for DNA sequencing using the M13 forwardprimer. Cy- Step Hold 1 Hold 2 Hold 3 Hold 4 cles 1 98° C./5 min — — — 12 98° C./30 sec 50° C./15 sec 60° C./4 min — 25 3 — — — 4° C./ — 99:99min

Sequencing reactions were transferred to a sterile eppendorf tube andadjusted to 20 μl total volume with ddH₂O, Sequencing products wereethanol precipitated by combining 20 μl of sequencing product with 16 μlof sterile water, and 64 μl of 95% non-denatured, ACS grade ethanol. Theprecipitation was incubated at room temperature for 15 min thencentrifuged at room temperature for 20 min at 12,000×g. The supernatantswere removed and discarded. The pellets were washed with 250 μl of 70%ethanol then centrifuged 10 min at 12,000×g. Sequencing products weredried in a heat block at 90° C. for 1 min then delivered for sequencingto Dr. Morgan V is of the Automatic DNA Sequencing Facility of theDepartment of Environment and Plant Biology, Ohio University.

DNA Sequencing of Synthetic Genes Using the EGFP566 as Primer

The plasmids pUC18-SStob-YK20-EGFP, pUC18-SStob-YK8-EGFPpUC18-SStob-YL20-EGFP, pUC18-SStob-YL8-EGFP, pUC18-SStob-FL8-EGFP, andpUC18-SStob-FK9-EGFP were sequenced from the 3′ end using the EGFP566 asprimer (FIG. 11; Integrated DNA Technologies) to verify in-frameligation of synthetic genes between SStob and EGFP. Sequencing reactionsusing the 566asEGFP primer were performed as described for the M13forward primer except the annealing temperature (Table 1-Step 2, Hold 2)was lowered to 47° C.

Transformation of Agrobacterium tumefaciens and Tobacco CellsTransformation of Agrobacterium tumefaciens

We transformed Agrobacterium tumefaciens (strain LBA4404) with plasmidspBI121-SStob-FK9-EGFP, pBI121-SStob-YK20-EGFP, pBI121-SStob-YK8-EGFP,pBI121-SStob-YL20-EGFP pBI121-SStob-YL8-EGFP, pBI121-SStob-FL8-EGFP, andpBI121-SStob-EGFP by the freeze-thaw method (An et al. 1-19). Theplasmids were isolated from LB liquid cultures (3-5 ml) supplementedwith kanamycin (30 μg/ml) using Wizard minipreps (Promega).Approximately 35-40 μl of plasmid was added directly to 100 μl ofcompetent, frozen Agrobacterium. The mixture was thawed, then quicklyfrozen in N₂ (I). The frozen tubes were heated to 37° C. for 5 min. LBwas then added to a final volume of 1 ml and the tubes were incubated at28° C. for 2-4 h with shaking at ˜100 rpm. The cells were then platedonto solid LB plates supplemented with kanamycin (30 μg/ml) andstreptomycin (40 μg/ml). The plates were incubated in the dark at 28° C.for 2-3 days. Positive transformants were picked and cultured in 3.5-5ml of LB medium having 30 μg/ml kanamycin and 40 μg/ml streptomycin at28° C. with gentle shaking at (100-175 rpm) for about 16-20 h. Thecultures were then pelleted by centrifugation (1000×g for 5-10 min) andthe supernatants removed and discarded. The pellets were resuspended in1 ml of fresh LB medium.

Transformation of Nicotiana tabacum Bright Yellow-2 (BY-2) SuspensionCultures

Tobacco cell-suspension cultures (Nicotiana tabacum, BY-2) weretransformed by Agrobacterium infection as previously detailed (McCormicket al. 81-84). Untransformed tobacco BY-2 cells were cultured (500 ml ina 1 L flask) for 4-5 days on a gyrotary shaker (88-94 rpm) at roomtemperature in NT-1 medium. The flask of cells was removed from theshaker and the cells were settled. Tobacco cells (10 ml) weretransferred under strerile conditions to empty Petri dishes. Fromsection 2.2.1, 100 μl of the transformed Agrobacterium suspension wasmixed with the cells. The cultures were parafilmed and incubated in thedark for 2-3 days at 28° C. Excess A. tumefaciens was removed from thecells by washing 3 times with 10-15 ml of sterile culture medium eitherNT-1 medium, Schenk and Hildebrandt medium (SH), or modified SH medium(SH*, SH sans kinentin). Washed tobacco cells were then plated on solidmedium (phytagel 2 g/L in either NT-1, SH, or SH*) supplemented withkanamycin (200 μg/ml) for selection and timentin (400 μg/ml) to kill theA. tumefaciens. Tobacco transformants appeared as small bumps on theplates after 3-5 weeks. These bumps were chosen for further propagationon kanamycin and timentin supplemented plates (2-4 generations) toensure complete eradication of the A. tumefaciens before the cells weretransferred to plates having only kanamycin.

Identification of Cell Lines Expressing Synthetic Genes and Localizationof Synthetic Gene Products Fluorescence Microscopy

Aliquots (5-10 ml) of suspension cultured cells were withdrawn directlyfrom liquid cell cultures (5-20 d). Expression of EGFP in transformedcell lines was viewed using a Zeiss LSM 510 confocal laser scanningmicroscope set for EGFP visualization (488 nm excitation; 510 nmemission).

Plasmolysis of Transformed and Untransformed Tobacco Cells forVisualization of EGFP by Fluorescence Microscopy

Suspension cultured YK20-EGFP, YK8-EGFP, YL8-EGFP, and FK9-EGFP, andSS-EGFP tobacco cells (5-20 d) were pipetted (10 ml) to 15 ml graduatedtubes. The cells were centrifuged and the packed cell volume wasrecorded. The cells were plasmolyzed by the addition of either of 1 Mpotassium phosphate buffer pH 7 to a final concentration of 500 mM or 1M mannitol to a final concentration of 750 mM. The plasmolyzed cellswere viewed by fluorescence microscopy.

Verification of Transgene Insertion by PCR

Isolation of Genomic DNA from Suspension-Cultured Tobacco Cells

Genomic DNA was prepared from tobacco suspension cultured cells usingthe DNAzol ES method (Molecular Research Center, Inc. Cincinnati, Ohio)according to manufacturer's instructions. Purity and concentration wereestimated by their 260/280 nm ratio and 260 nm absorbances respectively.

PCR Amplification of Transgenes from Genomic DNA

Insertion of our transgenes SStob-YK20-EGFP and SStob-YL8-EGFP intotobacco was verified by PCR amplification using SSeq4Sen (TaOpt=43° C.;Tm=68° C.) and EGFP566 as (TaOpt=47° C.; Tm=73.1° C.) primers (FIG. 11).Master mixes were created so that each reaction (25 μl) contained 12.5μl of 2× Promega PCR master mix (Promega), 0.125 nmol of SSeq4Senprimer, 0.125 nmol EGFP566 as primer, 10% (v/v) glycerol, and 0.14-0.21ng of genomic template DNA. Due to a high % GC content in both primerand template DNA, a preincubation step of 98° C. for 5 min was employed.The optimized PCR program is shown in Table 2. After completion of thePCR cycles, the reactions were separated by 1% agarose gelelectrophoresis, eluted from the gel, and DNA sequenced using theSSeq4Sen oligonucleotide primer.

TABLE 2 Thermocycler program for synthetic gene amplification by PCR.Step Hold 1 Hold 2 Hold 3 Cycles 1 98° C./5 min — — 1 2 98° C./30 sec55° C./1 min 74° C./3 min 25 3 — — 74° C./5 min 1 PCR amplicficationreactions were performed using primers SSeq4Sen and EGFP566as on anApplied Biosystems GeneAmp PCR system 2400.

Isolation and Purification of Synthetic Gene Products Liquid CellCulture

Transformed tobacco cells were cultured in 500 ml of SH* mediasupplemented with kanamycin (150 μg/ml) in 1 L Erlenmeyer flasks atambient temperatures on a gyrotary shaker set to 88-94 rpm. Cells weresubcultured every 14-21 days (FIG. 12).

Culture Medium Harvest

Liquid cell cultures (10-21 days) were filtered on a sintered glassfunnel (Pyrex 40-60 ASTM). The culture media was collected, concentratedby rotary evaporation to 50-250 ml, then dialyzed in 3,500 Da molecularweight cut-off (MWCO) dialysis tubing (Spectrum Laboratories, Inc.,Rancho Domingo, Calif.) against ddH₂O at 4° C. for 36 h.

Isolation of Fusion Glycoproteins by Hydrophobic InteractionChromatography

Dialyzed medium was re-concentrated by rotary evaporation to 50-250 ml,adjusted to 2 M NaCl (aq) then centrifuged at 20,000×g for 20 min.Supernatants were loaded onto a Butyl-Sepharose 4 Fast Flow hydrophobicinteraction chromatography (HIC) column (FIG. 12; 1.6 cm i.d.×40 cm,Amersham Pharmacia Biotech) equilibrated in 2 M NaCl (aq). Elutionproceeded stepwise from 2 M NaCl (aq) to 1 M NaCl (aq), and finally toddH₂O at flow rates of 1-2 ml/min. Visibly green fractions eluting in 1M NaCl were collected, dialyzed against ddH₂O (3,500 Da MWCO or 6-8,000Da MWCO dialysis tubing for 36 h at 4° C.; Spectrum Laboratories, Inc.)and then lyophilized.

Fractionation of Fusion Glycoproteins by Gel Filtration Chromatography

The HIC fractionated fusion glycoproteins were dissolved ˜30 mg/ml in200 mM sodium phosphate, 0.005% (w/v) sodium azide, pH 7 (Superosebuffer) then chromatographed on either a semi-preparative Superose-12column or Superose-6 gel filtration column (FIG. 12; 16 mm i.d.×500 mm,Amersham Pharmacia Biotech) equilibrated in Superose buffer, pH 7.Spectrophotometric detection was monitored at 220 nm. Flow rates werebetween 0.5-1 ml/min. Visibly green fractions corresponding to peaksabsorbing at 220 nm were pooled and either stored at −20° C. ordialyzed, lyophilized, then stored at −20° C.

Isolation of Fusion Glycoproteins by Reversed Phase HPLC

The fusion glycoproteins isolated after gel filtration chromatographytypically were directly injected onto a semi-preparative C4reversed-phase HPLC column (FIG. 12; 10 mm i.d.×250 mm, Vydac, Hesperia,Calif.) equilibrated in 0.1% (v/v) TFA (aq) and eluted with a lineargradient to 100% end buffer (0.1% (v/v) TFA (aq) in 80% (v/v)acetonitrile) at 2 ml/min over 60 min. UV detection was monitored at 220nm. The major peak absorbing at 220 nm corresponded to each fusionglycoprotein and was collected and lyophilized. A Hewlett-Packard series1050 HPLC was used for purification chromatography.

Removal of the EGFP Tag by Tryptic Digestion

Fusion glycoproteins (5-100 mg) were dissolved in water to 28.6 mg/mland heated to 100° C. for 2-3 min. Once cool, samples were diluted to 5mg/ml in buffer containing 10 mM CaCl₂ (aq) and 2% (w/v) NH₄HCO₃ (aq).Trypsin was added 100:1 (w/w) substrate to enzyme. Reactions wereincubated 16-24 h at room temperature with constant stirring. Thedigestion reactions were lyophilized, then fractionated by Superose-12gel filtration chromatography and C4 reversed phase chromatography asdescribed above. Peaks corresponding to YK20, YK8, YL8, and FK9glycomodules (designated YK20, YK8, YL8, and FK9, respectively) werecollected and lyophilized. The purified glycomodules were used for aminoacid composition analyses and as substrates for in vitro crosslinking.

Anhydrous Hydrogen Fluoride (HF) Deglycosylation of Fusion Glycoproteins

The dried fusion glycoproteins YK20-EGFP, YK8-EGFP, and YL8-EGFP (5-10mg) were deglycosylated with anhydrous HF as described earlier (Sangerand Lamport 66-70). Briefly, anhydrous HF containing 10% (v/v) anhydrousmethanol at 0° C. was added (2-10 mg/ml) to fusion glycoprotein samples.Reactions were incubated on ice for 1 h with constant stirring thenquenched by adding them to 6 volumes of ice-cold ddH₂O. Thedeglycosylated samples were dialyzed at 4° C. against ddH₂O, thenlyophilized. Dried, deglycosylated fusion glycoproteins designateddYK20-EGFP, dYK8-EGFP, and dYL8-EGFP were isolated by analytical C4reversed phase chromatography as described above for semi-preparative C4HPLC, except the gradient was eluted over 40 min.

Characterization of Transgene Products Amino Acid Composition Analyses

Glycomodules YK20, YK8, and YL8, and crosslinked YK20 (YK20XL) wereanalyzed for amino acid composition as described by (Bergman, Carlquist,and Jornvall 45-55). Briefly, samples were hydrolyzed at 110° C. for18-24 h in constant boiling 6 N HCl (aq) (Pierce, Rockford, Ill.)containing 10 mM phenol (Pierce). Hydrolyzed samples were cooled to roomtemperature then dried under a stream of N₂ (g). Hydrolysates (20 μg)were derivatized with phenylisothiocyanate (PITC) for 15-30 min to formphenylthiocarbamyl-amino acid (PTC-aa) derivatives. The PTC-aaderivatives were separated by reversed phase HPLC on a Prodigy ODS (3)C18 analytical column (4.6 mm i.d.×150 mm, 3 μm particle size, 10 nmpore size, Phenomenex, Torrance, Calif.) with UV detection at 254 nm.The IDT quantity was determined from the same hydrolysates using aseparate assay described earlier (Schnabelrauch et al. 477-89).

The amino acid composition of FK9 was determined by Dr. Joseph F. Leykamof the Macromolecular Facility at Michigan State University, EastLansing, Mich.

Partial N-Terminal Sequencing

Partial N-terminal sequences of the fusion glycoprotein FK9-EGFP andanhydrous HF deglycosylated fusion proteins dYK20-EGFP, dYK8-EGFP, anddYL8-EGFP were obtained by Joseph F. Leykam on a 477A AppliedBiosystems, Inc. gas-phase sequencer by automated Edman degradation.

Colorimetric Hydroxyproline Estimation

Samples (100-1000 μg) were hydrolyzed in 6 N HCl (aq) (not more than 4mg/ml) at 110° C. for 18-24 h. Hydrolysates were dried under N₂ (g) thenredissolved in 0.5 ml distilled deionized water. Hydroxyprolineestimation was determined by colorimetric assay as described earlier(Lamport and Miller 454-56; Lamport and Northcote 665-66).

Detection and Quantification of Isodityrosine (IDT)

Acid hydrolysates of YK20, YK8, YL8, FK9, and YK20XL were fractionated(10 μg each) on a PolyHYDROXYETHYL A column (9.4 mm i.d.×200 mm, 5 μmparticle size, 10 nm pore size, PolyLC Inc., Columbia, Md.) elutedisocratically in size exclusion mode (SEC) with 50 mM formic acid at 0.8ml/min. UV detection was monitored at 280 nm for tyrosine absorbance(Schnabelrauch et al. 477-89). IDT was partially identified byco-chromatography with an authentic external IDT standard (courtesy ofDr. Derek T. A. Lamport). A three level calibration was performed usingpeak areas of known amounts of the external IDT standard, from which anaverage molar response factor was calculated. This response factor wasused to quantify IDT from peak areas of our transgene products.

Hydroxyproline-O-Glycoside Profiles of the Transgene Products

We determined the Hyp-O-glycosylation profiles of YK20-EGFP, YK8-EGFP,YL8-EGFP, and FK9-EGFP as previously described (Lamport and Miller454-56). The fusion glycoproteins YK20-EGFP (2.20 mg), YK8-EGFP (4.10mg), YL8-EGFP (2.25 mg), and FK9-EGFP (2.21 mg) were each dissolved to 5mg/ml in 0.44 N NaOH (aq) and hydrolyzed at 105° C. for 18 h. Sampleswere cooled on ice, neutralized with ice-cold 1 M H₂SO₄ (aq), and thenlyophilized. Dried hydrolysates were redissolved in ddH₂O, loaded onto acation exchange column (6 mm i.d.×750 mm, Chromobeads C-2, Technicon)equilibrated in water, and eluted with a linear gradient to 0.5 M HCl(aq). An automated, in-line post-column hydroxyproline assay providedthe detection of Hyp-glycosides at 560 nm.

Neutral Sugar Composition Analyses

The neutral sugar compositions of YK20-EGFP, YK8-EGFP, YL8-EGFP,FK9-EGFP, and YK20XL (100 μg each) were determined as alditol acetatederivatives (Albersheim et al. 340-45) using myo-inositol (50 nmol) asan internal standard as previously described (Shpak, Leykam, andKieliszewski 14736-41). Briefly, samples containing internal standardwere hydrolyzed in 200 μl of 2 N trifluoroacetic acid at 121° C. for 1h. Hydrolysates were dried under N₂ (g) at 50° C. Aldehyde groups of thefree aldose sugars were reduced to aiditols with sodium borohydride(NaBH₄ 20 mg/ml in 3 M NH₄OH) at room temperature for 1 h. This reactionwas neutralized with glacial acetic acid, and dried thoroughly under N₂(g) at 40° C. then dessicated overnight. Samples were acetylated withacetic anhydride (121° C. for 1 h.) then separated by gas chromatography(GC). GC was performed on an HP 5890 Series II GC using an HP-5 column(Crosslinked 5% PH ME Siloxane; 30 m (L) X 0.32 mm (I.D.) X 0.25 μm filmthickness; Hewlett Packard, USA) programmed from 130° C. to 177° C. at arate of 1.2° C./min then from 177 to 200° C. at a rate of 10° C./min.

Uronic Acid Estimations

Uronic acids were detected by a manual colorimetric assay (Blumenkrantzand Asboe-Hansen 484-89). The glycomodules YK20, YK8, YL8, FK9, YK20XL(100 μg) were dissolved in concentrated sulfuric acid having 12.5 Msodium tetraborate, and then cooled on ice for 5 min. The samples werehydrolyzed at 100° C. for 5 min and again cooled on ice. Them-Hydroxydiphenyl reagent (150 mg m-hydroxydiphenyl in 100 ml 0.5% (w/v)NaOH) was reacted with the samples for 5 min at room temperature andthen the 520 nm absorbances were recorded. Uronic acid quantities wereestimated from the 520 nm absorbances of external galacturonic acidstandards.

Confirmation of Hyp-O-Arabinogalactan Polysaccharide in YL8-EGFP by GelFiltration of Base Hydrolysates

To confirm that YL8-EGFP contained O-linked Hyp arabinogalactanpolysaccharide (Hyp-PS), 10.9 mg of reverse-phase purified YL8-EGFP wasdissolved 5 mg/ml in 0.44N NaOH (aq) and hydrolyzed at 105° C. for 18 h.This cleaves the peptide backbone, but leaves the Hyp-O-linkedglycosides intact. The hydrolysate was cooled on ice, neutralized with 1NH₂SO₄ (aq) to pH 8.3, then lyophilized. The dried hydrolysate wasdissolved in 1 ml of 20% (v/v) acetonitrile (aq) and then centrifuged 20min at 10,300×g. Supernatants (500 μl) were separated on a Superdex-75gel filtration column equilibrated and eluted isocratically (0.4 ml/min)in 20% (v/v) acetonitrile (aq). Fractions were collected (1min/fraction), lyophilized, and then analyzed for hydroxyproline content(Lamport and Miller 454-56; Lamport and Northcote 665-66).

In Vitro Crosslinking of Extensin Precursors

Isolation of the pl 4.6 Extensin Peroxidase from Tomato

Untransformed Bonnie Best tomato cell suspension cultures (500 ml) weregrown 10-30 days in SH* medium. The spent culture medium was filtered onsintered glass funnels, immediately shell frozen (350 ml) andlyophilized. Dry culture medium was redissolved in ice-cold ddH₂O to 50ml and ultra-centrifuged at 250,000×g (60,000 rpm) for 4 h. at 10° C. toremove pectin contaminants. Supernatants were adjusted to 95% saturationwith ammonium sulfate (s), gently stirred at 4° C. for 16-20 h, and thencentrifuged at 27,000×g for 20 min. The pellets were dissolved in anddialyzed against 4×1 L volumes of ice-cold 20 mM sodium acetate bufferpH 6 for 18-24 h. at 4° C. in Spectra-Por 3,500 Da MWCO dialysis tubing(Spectrum Laboratories Inc.). Samples were centrifuged at 12,100×g for15 min at 4° C. Supernatants were injected onto a preparative DEAESepharose Fast Flow (Amersham Pharmacia) anion exchange column (14 mmi.d.×9.6 cm) equilibrated in 20 mM sodium acetate buffer, pH 6 andeluted at a flow rate of 1 ml/min for 1 h. A linear gradient to 2M NaClin 20 mM sodium acetate buffer, pH 6 was then applied by a gradientmaker. Fractions (1 ml) were collected and assayed for peroxidaseactivity by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)assay as described earlier (Everdeen et al. 616-21). Active fractionswere pooled and concentrated by Centricon YM-30 centrifugal filterdevices (Millipore, USA). Retentates were pooled, adjusted to 10% (v/v)glycerol, frozen with N₂ (I) in 100-250 μl aliquots, and stored at(−80)° C.

Purification of pl 4.6 Extensin Peroxidase from Tomato by HPLC

Anionic peroxidase fractions from preparative DEAE anion exchangechromatography were further separated by normal-phase ion exchange HPLCon a DEAE-5PW column (Tosohaas, 7.5 mm i.d.×750 mm). The column wasequilibrated in 20 mM sodium acetate pH 6 and eluted by a lineargradient to 500 mM NaCl (aq) in 20 mM sodium acetate pH 6 over 100minutes with a flow rate of 1 ml/min. Heme group absorbance, typical ofclass III plant peroxidases (Welinder et al. 6063-81), was monitored at405 nm.

Quantification of Peroxidase Activity by2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic Acid) (ABTS) Assay

Peroxidase activity was quantified by ABTS assay as described earlier(Everdeen et al. 616-21; Schnabelrauch et al. 477-89), whereby 12 ng ofperoxidase will produce an absorbance change of 1 A_(405 nm) unit perminute.

In Vitro Crosslinking of P3 Glycomodules with Tomato Extensin Peroxidase

Tomato P1 extensin was isolated as described earlier for use as apositive crosslinking control (Smith et al. 1021-30; Smith, Muldoon, andLamport 1233-39). Reversed phase HPLC purified glycomodules YK20, YK8,YL8, and FK9 and native tomato P1 extensin were used as substrates forin vitro crosslinking reactions. Extensin substrate stock solutions (6mg/ml) were prepared by diluting 10 mg/ml substrates in ddH₂O withMcIlvaine buffer pH6. Crosslinking reactions were performed at roomtemperature by combining substrate (3 mg/ml), extensin peroxidase enzyme(1 ng per 60 μg of substrate), and H₂O₂ (60 μM). Reaction time wasinitiated upon addition of H₂O₂. The stopping reagent (50 mM2-mercaptoethanol) was added prior to H₂O₂ in time zero reactions toprevent crosslinking from occurring. Reactions were stopped by theaddition of 50 mM 2-mercaptoethanol (16.7 mM final concentration) afterspecified times.

Detection of in vitro crosslinking and rate estimations were provided byanalytical Superose-6 gel filtration chromatography eluted isocraticallyin pH 7 Superose buffer (Amersham Pharmacia; 0.75 ml/min; OD 220 nmdetection). Crosslinking rates were calculated by the decrease in peakarea of the monomer after 1 min of incubation versus an equal amount ofthe time zero reaction using the first order rate equation(Schnabelrauch et al. 477-89). Rates were expressed as the amount ofsubstrate crosslinked (μg) per sec (Schnabelrauch et al. 477-89).

Detection of Extensin Peroxidase Activity in Tobacco Cell SuspensionCultures

Untransformed tobacco BY-2 suspension cultured cells were grown 10 daysin NT-1 culture medium to 17% PCV. The cells (100 ml) were filtered on asintered glass funnel, washed with 1.25 L of water, and then eluted with500 ml of 1 M KCl (aq) in 20 mM sodium acetate buffer, pH 6. Thissalt-eluted fraction was designated (E2). Spent culture medium (350 ml)was lyophilized then redissolved to 50 ml with ice-cold water. Thiscrude medium fraction was designated (E1). Subsequent steps were kept ator below 4° C. The salt-eluate E2 sample was then dialyzed (24-36 h; 4°C.; 3,500 Da MWCO tubing) against 20 mM sodium acetate buffer, pH 6. A250 ml aliquot of the dialyzed E2 was lyophilized, and then redissolvedto 1 ml with water. Enzyme activity was estimated by ABTS assay. The E1and E2 samples were then examined for extensin crosslinking.

In Vitro Crosslinking with Crude Cationic Tomato Peroxidases

Crude tomato culture medium was prepared as described for pl 4.6extensin peroxidase preparation. The preparative DEAE void fraction(non-binding) was collected. An aliquot (10-15 ml) was concentrated to3.2 ml using Centricon YM-30 centrifugal filter devices (Millipore). Theconcentrated void peroxidases were adjusted to 10% (v/v) glycerol andthen stored at −80° C. The void peroxidases were quantified by ABTSassay then tested for in vitro crosslinking. Crosslinking reactions wereincubated with substrates (3 mg/ml), enzyme (1.7 ng/60 μg substrate),and H₂O₂ (60 μM) for 0 and 30 min. The reactions were separated by gelfiltration on an analytical Superose-6 column (Amersham Pharmacia)eluted at 0.5 ml/min with UV detection at 220 nm.

Analysis of Crosslinked Extensin Products

Isolation of Diisodityrosine (di-IDT) From Untransformed Tomato CellWalls

Isolation of Cell Walls

Untransformed Bonnie Best tomato cell suspension cultures were filteredon a sintered glass funnel. The cell walls were isolated as describedearlier (Lamport 151-218; Lamport and Northcote 52P).

Acid Hydrolysis of Untransformed Tomato Cell Walls

In a closed reflux apparatus, 1.55 g of untransformed tomato cell wallswere added to 465 ml of 6 N HCl (aq) containing 10 mM phenol (Pierce).The hydrolysis vessel was purged of air by vacuum then flushed with N₂(g). The cell walls were refluxed for 24 h then cooled. The hydrolysatewas concentrated by rotary evaporation then dried completely under astream of N₂ (g).

Strong Cation Exchange Chromatography of Cell Wall Hydrolysates

The cell wall hydrolysate was redissolved in 100 ml of ddH₂O thenchromatographed on Whatman P11 strong cation exchange column eluted bygravity as described earlier (Brady, Sadler, and Fry 323-27). Fractions0-9 were collected (100 ml each) and concentrated to dryness by rotaryevaporation. Fractions 0-9 were each redissolved in 1 ml of ddH₂O. A 1μl aliquot of fractions 0-9 was spotted on Whatman 3 mM chromatographypaper and examined under UV light (254 nm). The spots were then exposedto NH₄OH vapor and reexamined under 254 nm light.

Paper Chromatography

Paper chromatography was performed as previously described (Brady,Sadler, and Fry 323-27). An authentic dilDT standard was a gift from Dr.Stephen C. Fry of the University of Edinburgh and was chromatographed todetect dilDT in fraction 7.

Size Exclusion Chromatography

Fraction 7 (500 μl) from P11 cation exchange chromatography was filteredwith Millipore spin filters (0.45 μm pore size; filter type HV; NihonMillipore Kogyo K.K. Japan). The filtrate (250 μl per injection) wasfractionated on a PolyHYDROXYETHYL A column (9.4 mm i.d.×200 mm; 5 μmparticle size, 20 nm pore size, PolyLC Inc.) eluted isocratically insize exclusion mode (SEC) with 50 mM formic acid at 0.8 ml/min and UVdetection at 280 nm (Schnabelrauch et al. 477-89). Six fractions,designated SEC1 F1-6, were collected, checked for blue fluorescence asdescribed in section 2.7.1.3, and then lyophilized. SEC1 F4 wasredissolved in 0.1 ml of ddH₂O and refractionated by SEC on aPolyHYDROXYETHYL A column (9.4 mm i.d.×200 mm; 5 μm particle size, 10 nmpore size, PolyLC Inc.; 10 μl per injection) as described above(Schnabelrauch et al. 477-89). Five fractions, designated SEC2 F1-5,were collected, checked for blue fluorescence, and then lyophilized.

Reverse-Phase HPLC Purification of Diisodityrosine

Diisodityrosine (my dilDT) was purified from SEC2 F4 by reversed phasechromatography on a Waters Spherisorb ODS-2 C18 column (4.6 mm i.d.×250mm; 5 μm particle size; Alltech Associates, Inc., Deerfield, Ill.).Briefly, SEC2 F4 was redissolved in 50 μl of ddH₂O and injected onto theC18 column equilibrated in ddH₂O. The column was washed with water for15 min then eluted at 0.8 ml/min with a linear gradient to 60% (v/v)acetonitrile (aq) over 25 min. A peak absorbing at 280 nm eluted at 24.8min was collected. Further fractionation of this peak on the 10 nm poresize PolyhydroxyethylA column confirmed its retention time at 10.4 min.

Isolation of YK20 Crosslinking Amino Acids In Vitro Crosslinking of YK20for Isolation of Crosslinking Amino Acids

YK20 (20 mg) was crosslinked overnight under the conditions described insection 2.6.4 with pl 4.6 extensin peroxidase isolated from tomatosuspension cultures. The reaction was stopped with 50 mM2-mercaptoethanol then lyophilized. The dried crosslinking reaction wasdissolved in a minimal volume of water then centrifuged at 10,300×g for20 min. The insoluble pellet (YK20XL) was washed with ddH₂O until theconductivity of the supernatants was equal to that of water.

Acid Hydrolysis of YK20XL and YK20 for the Isolation of CrosslinkingAmino Acids

The YK20XL and YK20 samples (2.2 mg and 1 mg respectively) weredissolved to 2 mg/ml in 6N HCl with 10 mM phenol in sealed glass conicalvials and then hydrolyzed for 20 h. at 110° C. in a heating block.Hydrolysates were dried under N₂ (g) then resuspended in 100 μl ddH₂O,

Size Exclusion Chromatography for the Purification of the CrosslinkingAmino Acids

Hydrolysates of YK20XL and YK20 were fractionated by SEC on aPolyHYDROXYETHYL A column (9.4 mm id X 200 mm; 5 μm particle size; 10 nmpore size; PolyLC) as previously described. UV detection was monitoredat 280 nm for tyrosine (and tyrosine derivative) absorbance. The peakswere collected and lyophilized.

Molecular Mass Determination of the YK20 Crosslinking Amino Acid byMALDI TOF Mass Spectrometry

The unknown crosslinking amino acid candidate was identified byMALDI-TOF mass spectrometry by Dr. Michael Hare at Oregon StateUniversity.

Identification of the Crosslinking Amino Acid Unknown by 1-D ¹HNMR

The unknown crosslinking amino acid from hydrolysate of YK20XL wascharacterized by 1-D ¹HNMR techniques by Dr. Li Tan of Ohio University.

Results Synthetic Gene Construction

The synthetic genes SStob-YK20-EGFP, SStob-YK8-EGFP, SStob-YL20-EGFP,SStob-YL8-EGFP, SStob-FK9-EGFP, and SStob-FL8-EGFP were assembled fromoligonucleotide primers and sequenced (Lewis et al. 400-06; Shpak 179;Shpak, Leykam, and Kieliszewski 14736-41; Tan L 1362-69) (FIG. 13). Thesynthetic gene SStob-EGFP was kindly donated by Dr. Elena Shpak. Thesesynthetic genes were inserted into the binary plant transformationvector pBI121 under the promotion of the CaMV 35 S constitutive promoterforming plasmids pBI121-SStob-YK20-EGFP, pBI121-SStob-YK8-EGFP,pBI121-SStob-YL20-EGFP, pBI121-SStob-YL8-EGFP, pBI121-SStob-FK9-EGFP,pBI121-SStob-FL8-EGFP, and pBI121-SStob-EGFP (Shpak, Leykam, andKieliszewski 14736-41).

Transformation of A. tumefaciens and Tobacco Cells with Synthetic Genes

Agrobacterium tumefaciens was transformed with plasmidspBI121-SStob-YK8-EGFP, pBI121-SStob-YK20-EGFP, pBI121-SStob-YL8-EGFP,pBI121-SStob-YL20-EGFP, pBI121-SStob-FL8-EGFP, pBI121-SStob-FK9-EGFP,and pBI-SStob-EGFP (McCormick et al. 81-84). Tobacco BY-2 cellsuspension cultures were subsequently transformed with these syntheticgenes by A. tumefaciens infection yielding cell lines designatedNtYK20-EGFP, NtYK8-EGFP, NtYL20-EGFP, NtYL8-EGFP, NtFK9-EGFP,NtFL8-EGFP, and NtSS-EGFP.

Verification of Synthetic Gene Insertion by PCR

Insertion of the synthetic genes SStob-YK20-EGFP and SStob-YL8-EGFP intotobacco cells was verified by PCR of genomic DNA with oligonucleotideprimers complimentary to SStob and EGFP (FIG. 14). Synthetic geneexpression of YK20-EGFP, YK8-EGFP, YL8-EGFP, and FK9-EGFP

The transformed cell lines NtYK20-EGFP, NtYK8-EGFP, NtYL8-EGFP,NtFK9-EGFP, NtSS-EGFP demonstrated EGFP expression when viewed byfluorescence microscopy (FIG. 15). Untransformed tobacco (uTob) cellswere viewed as a control. Although NtSS-EGFP and NtYL20-EGFPdemonstratedEGFP expression when viewed by fluorescence microscopy (not shown), nofusion glycoprotein was able to be isolated from the culture medium ofthese cell lines. The FL8-EGFP cell line was never observed to exhibitgreen fluorescence (not shown).

Cellular Localization of YK20-EGFP to the Cell Wall

The YK20-EGFP, SS-EGFP, and uTob cells were plasmolyzed to determine thecellular localization of their respective transgene products. Thefluorescence in YK20-EGFP cells was localized to the cell wall, with aconcentration in the crosswall, whereas SS-EGFP was localized tointracellular compartments (FIG. 16). Plasmolysis of YK8-EGFP, YL8-EGFP,or FK9-EGFP cells did not demonstrate visible fluorescence in the cellwall (not shown). The YK20-EGFP fusion proteins remained with the cellwall after plasmolysis with 500 mM potassium phosphate pH 7 (FIG. 16E).

Isolation of the Fusion Glycoproteins YK20-EGFP, YK8-EGFP, YL8-EGFP andFK9-EGFP

Transformed YK20-EGFP, YK8-EGFP, YL8-EGFP, and FK9-EGFP cellsdemonstrated EGFP secretion into the culture medium. Concentratedculture medium was fractionated by HIC (FIG. 17). Fluorescent greenfractions were collected, desalted by dialysis, and then lyophilized.

To further purify the HIC-fractionated YK20-EGFP, YK8-EGFP, YL8-EGFP,and FK9-EGFP fusion glycoproteins, gel filtration chromatography wasperformed using either a Superose-6 or 12 column. Chromatographstypically showed two fluorescent green peaks absorbing at 220 nm. Thelarger molecular weight peak corresponded to the P3-EGFP fusionglycoprotein and the smaller for cleaved EGFP (not shown).

The YK20-EGFP, YK8-EGFP, YL8-EGFP, and FK9-EGFP fusion glycoproteinseluted between 55-65% end buffer as single peaks upon C4 reversed phaseHPLC fractionation (FIG. 18). Typically, the fusion glycoprotein yieldsranged from 3-27 mg/l medium for YK20-EGFP, from 4-7 mg/l for YK8-EGFP,and from 6-23 mg/l for YL8-EGFP. Yields for FK9-EGFP were lower at 0.1to 3.3 mg/l.

Isolation of the Glycomodules YK20, YK8, YL8, and FK9

Fractionation of tryptic digestions of YK20-EGFP, YK8-EGFP, YL8-EGFP andFK9-EGFP by gel filtration followed by reversed phase HPLC yielded pureglycomodules designated YK20, YK8, YL8, and FK9 (FIG. 19). Trypsin wasnot able to cleave lysine residues within the P3 modules, presumably dueto the protection of the peptide backbone by the Hyp-O-oligoarabinosides(Lamport 1155-63; Smith et al. 1021-30; Stafstrom and Staehelin 242-46).

Deglycosylation of the Fusion Glycoproteins YK20-EGFP, YK8-EGFP, andYL8-EGFP by Anhydrous Hydrogen Fluoride Solvolysis

Deglycosylated fusion proteins were purified by C4 reversed phase HPLC(FIG. 20). Protein weight recoveries were recorded to estimate the massof carbohydrate removed (Table 3). FK9-EGFP was not deglycosylated byanhydrous HF solvolysis.

TABLE 3 Estimated percent weight carbohydrate of YK20-EGFP, YK8-EGFP,and YL8-EGFP. Sample Protein wt. recovery (%) Carbohydrate wt. recovery(%) YK20-EGFP 42 68 YK8-EGFP 49 51 YL8-EGFP 48 52 Protein weightrecoveries were recorded after HF deglycosylation, from which thepercent carbohydrate was estimated.

Amino Acid Composition Analyses of the YK20, YK8, YL8, and FK9Glycomodules

Amino acid compositions of the purified glycomodules YK20, YK8, YL8 andFK9 were determined (Table 4). All of the samples exhibited nearlycomplete hydroxylation of proline residues. Isodityrosine (IDT), adiphenyl ether-linked tyrosine dimer (FIG. 1) was observed in thehydrolysates of YK20, YK8, and YL8, but not in FK9 (FIG. 21).

TABLE 4 Amino acid compositions of the glycomodules YK20, YK8, YL8, andFK9. Amino Acid Composition (mol %) YK20 YK8 YL8 FK9 Amino Pro- Pro-Pro- Pro- Acid tein Gene tein Gene tein Gene tein Gene Hyp 59.8 0 60 056 0 52.5 0 Pro 0 55.4 0 54.1 0 54.1 0 55.3 Ser 18.2 19 18 19.3 20 19.317.8 19.5 Tyr 9 18.3 9 17.8 13 17.8 0 0 1/2IDT 8.7 0 8 0 5 0 0 0 Phe 0 00 0 0 0 21 17.5 Lys 4.3 6.4 5 6.7 0 0.7 7 6.5 Leu 0 0 0 0 6 6 0.6 0 Thr0 0.3 0 0.7 0 0.7 0 0 Met 0 0.3 0 0.7 0 0.7 0.9 0.6 Val 0 0.3 0 0.7 00.7 0.2 0.6 Total 100 100 100 100 100 100 100 100 Actual compositions(Protein) are compared to the deduced compositions (Gene) from syntheticgene translations.Partial N-Terminal Sequencing of dYK20-EGFP, dYK8-EGFP, dYL8-EGFP andFK9-EGFP by Automated Edman Degradation

The partial N-terminal sequences of dYK20-EGFP, dYK8-EGFP, dYL8-EGFP andFK9-EGFP matched those predicted from the gene sequences (Table 5). Thetwo terminal Arg residues of dYK20-EGFP, dYK8-EGFP, and dYL8-EGFP fusionproteins were a result of restriction site design. Originally, YK20-EGFPand YL8-EGFP were sent for sequencing, but the sequences did not exactlymatch those predicted from their genes. Deglycosylation of these fusionglycoproteins prior to N-terminal sequencing, however, yielded theirpredicted sequences.

TABLE 5 Partial N-terminal sequences of dYK20-EGFP, dYK8-EGFP,dYL8-EGFP, and FK9-EGFP. Fusion protein Partial N-terminal sequencedYK20-EGFP RRPSOOOOYYYKSOOOOSOS dYK8-EGFP RROSOOOOYYYKSOOOOSOS dYL8-EGFPRRPSOOOOYYYLSOOOOSOS FK9-EGFP SOOOOSOSOOOOFFFKSOOOOSOSOOOO

Hyp-O-Glycoside Profiles of the YK20-EGFP, YK8-EGFP, YL8-EGFP andFK9-EGFP Fusion Glycoproteins

All of the fusion glycoproteins showed greater than 82% of the total Hypresidues possessed O-linked tri or tetraoligoarabinosides (Table 6) aspredicted by the Hyp Contiguity Hypothesis (Kieliszewski and Lamport157-72). With the exception of YL8-EGFP, Hyp-PS was not detected in thefusion glycoproteins.

TABLE 6 Hyp-O-glycoside profiles of the YK20-EFGP, YK8-EFGP, YL8-EGFPand FK9-EGFP fusion glycoproteins. Hyp- Percentage of totalhydroxyproline Glycoside^(a) YK20-EGFP YK8-EGFP YL8-EGFP FK9-EGFP Hyp-PS0 0 3 0 Hyp-Ara₄ 56 56 55 42 Hyp-Ara₃ 32 31 27 40 Hyp-Ara₂ 4 4 6 5Hyp-Ara₁ 5 5 4 5 NG-Hyp 3 4 5 8 EGFP does not contain Hyp and is notglycosylated (Shpak E. et al. 1999). ^(a)Hyp-PS, Hyp polysaccharide;Hyp-Ara_(n), Hyp-arabinoside₁₋₄; NG-Hyp, non-glycosylated Hyp.

Neutral Sugar Composition Analyses of the Fusion GlycoproteinsYK20-EGFP, YK8-EGFP, YL8-EGFP, and FK9-EGFP

At least 84% of the neutral sugar carbohydrate attached to our fusionglycoproteins was arabinose (Table 7). Significant amounts of galactosewere observed in all of the fusion glycoproteins indicating the presenceof monogalactosyl-serine (Ser-O-gal). A lower ratio ofarabinose:galactose was observed for YL8-EGFP. Essentially no uronicacids were detected in any of the fusion glycoproteins or glycomodules.Thus, we estimated that YK20-EGFP was 39% wt carbohydrate (CHO),YK8-EGFP was 48% wt. CHO, YL8-EGFP was 45% wt. CHO, and FK9-EGFP was 32%wt. CHO.

TABLE 7 Neutral sugar compositions of the YK20-EGFP, YK8-EGFP, YL8-EGFP,and FK9-EGFP fusion glycoproteins. Glycosyl Glycosyl Composition (mol %)residue YK20-EGFP YK8-EGFP YL8-EGFP FK9-EGFP Arabinose 90 91 84 91Galactose 8 7 12 9 Rhamnose 1 1 2 0 Xylose 0 0 1 0 Glucose 1 1 1 0 EGFPis not glycosylated (Shpak, E. et al 1999).

Detection of Hyp-PS in the YL8-EGFP Fusion Glycoprotein

Base hydrolysis followed by gel filtration chromatography indicated ahigh molecular wt. (HMW) Hyp-O-glycoside occurred in YL8-EGFP andcomprised approximately 5.2% of the total Hyp as compared to 3% of thetotal Hyp calculated from the Hyp-O-glycoside profile (FIG. 22). Thisconfirmed that YL8-EGFP contains a small amount of Hyp-PS.

Estimated Molecular Mass of the Fusion Glycoproteins YK20-EGFP,YK8-EGFP, YL8-EGFP, and FK9-EGFP

Molecular masses of YK20-EGFP, YK8-EGFP, YL8-EGFP, and FK9-EGFP wereestimated from the Hyp-O-glycoside data, the neutral sugar compositiondata, and the deduced amino acid composition data. EGFP contributes26,924 Da to each fusion glycoprotein. The weight contributions of thearabinooligosaccharides were estimated from the Hyp-O-glycoside profiles(Table 8). Galactose was estimated from the neutral sugar compositionsand the weight contribution of the arabinooligosaccharides (Table 9).The protein weight contributions of the modules were predicted from thededuced amino acid sequence (Table 10; as shown in FIG. 13). The sum ofthese values yielded predicted molecular weights of each fusionglycoprotein (Table 11).

TABLE 8 Weight contribution of the arabinooligosaccharides of YK20-EGFP.MW of YK20-EGFP % of Hyp Number/molecule Arabinosides Daltons Hyp-(Ara)₄56 101 528 53222 Hyp-(Ara)₃ 32 58 396 22810 Hyp-(Ara)₂ 4 7 264 1901Hyp-(Ara)₁ 5 9 132 1188 NG-Hyp 3 5 0 0 Totals 100 180 79121 Weightcontributions of the O-linked arabinooligosaccharides were estimatedfrom the Hyp-O-glycoside profile data. Reported here is YK20-EGFP only.The YK8-EGFP, YL8-EGFP, and FK9-EGFP fusion glycoprpteins were estimatedthe same way. MW, molecular weight (Da/mol)

TABLE 9 Weight contribution of the monogalactosides of YK20-EGFP.YK20-EGFP Number/Molecule MW of Gal Daltons of Gal Ser-O-Gal 53 162.28642 NG-Ser 8 0 0 Total Ser 61 8642 From table 6 we know that the weightcontribution of the oligoarabinosides is 79121. From table 5 we knowthat the fusion glycoprotein is 90 mol % arabinose thus (599 molequiv.). Galactose was 8 mol % galactose (53 mol equiv.) The weightcontribution of galactose was calculated from these values. The weightcontributions of galactosides in YK8-EGFP, YL8-EGFP, and FK9-EGFP fusionglycoproteins were estimated the same way. MW, molecular weight(Da/mol); NG-Ser, non-glycosylated Ser.

TABLE 10 Weight contribution of the protein of YK20. Number of Aminoacid residues Residue wt. Daltons Module mol % Ser 61 87.08 5312 18.8Hyp 180 113.10 20358 55.4 Tyr 60 147.18 8831 18.5 Lys 20 128.17 2563 6.2Pro 1 97.12 97 0.3 Thr 1 101.11 101 0.3 Arg 2 156.19 312 0.6 Total 32537575 100.0 The deduced translation of the YK20 gene sequence in FIG. 1Awas used to estimate the molecular wt. contribution of the polypeptide.The weight contributions of the polypeptide in YK8-EGFP, YL8-EGFP, andFK9-EGFP were estimated the same way.

TABLE 11 Estimated molecular weights (Da) of YK20-EGFP, YK8-EGFP,YL8-EGFP, and FK9-EGFP. Weight Contributors YK20-EGFP YK8-EGFP YL8-EGFPFK9-EGFP Arabinosides 79121 31152 30096 32208 Galactosides 8642 29455283 3914 EGFP 26924 26924 26924 26924 Module 37575 15387 15267 17237Total (Daltons) 152262 76408 77570 80283 Summation of the wtcontributions of the arabinosides, the galactosides and the polypeptidecomponents yielded the estimated molecular weights of each fusionglycoprotein. Values are reported in Daltons.

These calculations indicate that YK20-EGFP is 152 kDa of which 58% iscarbohydrate (CHO), YK8-EGFP is 76.4 kDa (45% wt. CHO), YL8-EGFP is 77.6kDa (45% wt. CHO), and FK9-EGFP is 80 kDa (45% wt. CHO). These valuescompare well with the weight recoveries after HF deglycosylation (Table3) and the neutral sugar compositions (Table 7), with the exception ofYK20-EGFP and FK9-EGFP. The weight percent carbohydrate of these fusionglycoproteins appear to be underestimated from the neutral sugarcompositions. Only YL8-EGFP has a Gal:Ser ratio>1, as some of the Galresidues are in a Hyp-PS linkage.

Isolation of pl 4.6 Extensin Peroxidase from Tomato

A single extensin peroxidase peak was retained and then eluted from theDEAE-5PW anion exchange HPLC column (FIG. 23). Thus, preparative DEAEpurified enzyme was used for all further crosslinking reactions.

In Vitro Crosslinking of Extensin Precursors with pl 4.6 ExtensinPeroxidase

Extensin peroxidase isolated from tomato cell suspension cultures wasused to determine the substrate requirements for intermolecular covalentcrosslinking in vitro. Native tomato P1 extensin was used as a positivecrosslinking control substrate (FIG. 24) (Schnabelrauch et al. 477-89).Native tomato P1, YK20, YK8, and YL8 were able to be covalentlycrosslinked in vitro by the tomato pl 4.6 extensin peroxidase (FIG. 25).The FK9 glycomodule was not a substrate for the pl 4.6 extensinperoxidase nor was BSA (FIGS. 26 and 27). It was also shown that boththe enzyme and peroxide were required for in vitro crosslinking of YK20(FIGS. 28 and 29). Peaks eluting after 22 min are a result of thecrosslinking buffers and stopping reagent (FIG. 30).

In Vitro Crosslinking Rate Determination for the P1, YK20, YK8, YL8 andFK9 Substrates

In vitro crosslinking reaction rates were determined from freshlyprepared substrate stocks of P1, YK20, YK8, and YL8 using a first orderrate equation. Crosslinking reactions were performed in triplicate andthe mean crosslinking rates (μg crosslinked per sec)+/−the estimatedstandard deviations were 6.2+/−0.68 for P1, 4.9+/−0.31 for YK20,3.4+/−0.13 for YK8, 1.4+/−0.07 for YL8 (FIG. 31). Since FK9 does notcrosslink, the reaction rate is reported as zero.

In Vitro Crosslinking of Extensin Precursors with Tobacco Salt-Eluates

To check for the presence of an extensin peroxidase in tobacco BY-2 cellsuspension cultures, in vitro crosslinking assays using crude tobaccoculture media (E1) and a salt-eluate of intact tobacco cells (E2) onnative tomato P1 extensin and YK20 substrates. The amount of enzymeactivity was quantified by ABTS assay for E1 and E2 and equal amounts ofboth enzymes (˜35 ng, 7 times the normal amount) were used per standardamount of substrate (60 μg). I found that the E2 was able to catalyzethe complete in vitro crosslinking of P1 and YK20 over a reaction timeof 18 h, whereas E1 was not.

In Vitro Crosslinking of YK20 with Crude Cationic Tomato Peroxidases

The preparative DEAE void contained a significant amount of peroxidaseactivity. This fraction was tested for its ability to crosslink P1,YK20, and YL8 in vitro. The crosslinking reactions were the same as thestandard reactions except, the enzyme concentration and the incubationtimes were doubled. A minor amount of crosslinking was observed for allthree substrates (FIG. 32).

Amino Acid Composition of the Crosslinked YK20

Amino acid compositions of the crosslinked YK20 (YK20XL) were comparedwith the YK20 glycomodule (Table 12). The amino acid compositions showeda decrease in IDT and total Tyr equivalents after crosslinking.

TABLE 12 Amino acid compositions of YK20 and YK20XL. Amino AcidComposition (mol %) YK20 YK20XL Amino Acid Protein Gene Protein Hyp 59.80 56.8 Pro 0 55.4 2.3 Ser 18.2 19 18.2 Tyr 9 18.3 11.3 1/2IDT 8.7 0 2.6Phe 0 0 0 Lys 4.3 6.4 6.4 Leu 0 0 0 Thr 0 0.3 2.4 Met 0 0.3 0 Val 0 0.30 Total 100 100 100 Losses of IDT and net Tyr equivalents were observedafter in vitro crosslinking.

Neutral Sugar Composition of the Crosslinked YK20

There were no significant differences in the neutral sugar compositionbetween the soluble YK20XL and the YK20 glycomodule (Table 13). NoteGlc, Xyl, and Rha are most likely contaminants. We did not detect uronicacids in either YK20 or YK20XL.

TABLE 13 Neutral sugar compositions of YK20 and YK20XL. Mol PercentGlycosyl Residue YK20 YK20XL Ara 91 88 Gal 7 9 Rha 1 0 Xyl 0 1 Glc 1 2 Aslight decrease in the mol % of arabinose and increase in galactose wereobserved upon crosslinking.Isolation of a Putative Crosslinking Amino Acid from Crosslinked YK20

A putative crosslinking amino acid, designated “unknown”, was detectedin the acid hydrolysates of YK20XL, but not in the YK20 glycomodule(FIG. 33). The unknown was collected and then fractionated again by sizeexclusion chromatography.

Identification of Crosslinking Amino Acid (Unknown) by PaperChromatography

Paper chromatography of the “unknown” compound from FIG. 33 and anauthentic diisodityrosine standard (Fry's dilDT) demonstrated that theyhad the same Rf value (Table 14). A putative dilDT standard (my dilDT)freshly prepared from tomato cell wall hydrolysates, alsoco-chromatographed with the unknown and the authentic standard.

TABLE 14 Paper chromatography of Fry's dilDT, “unknown”, and my dilDT inBAW. Sample Rf value Fluorescence* Fry's dilDT 0.06 + Unknown 0.06 + MydilDT 0.06 + *Vivid blue fluorescence was observed when viewed undershort-wave (254 nm) UV light after exposure with NH₃ vapor.

Identification of Crosslinking Amino Acid (Unknown) by Size ExclusionChromatography

The Fry's dilDT standard and the unknown were fractionated by sizeexclusion chromatography. The authentic dilDT standard co-eluted withthe unknown (not shown).

Identification of Crosslinking Amino Acid (Unknown) MALDI-TOF MassSpectrometry

The unknown was sent to Dr. Michael Hare of Oregon State University formolecular weight determination by MALDI-TOF mass spectrometry. Thetheoretical molecular mass of dilDT is [M+H]=719. The mass spectrum ofthe unknown peak contained a molecular ion of [M+H]=719. The samemolecular ion was observed in Fry's dilDT both here and as reportedearlier (Brady, Sadler, and Fry 323-27) and my dilDT standard. Thus, theputative crosslinking amino acid formed in vitro was dilDT.

Identification of Crosslinking Amino Acid (Unknown) 1-D ¹HNMR

The 1-D ¹HNMR spectrum of the “unknown” sample showed 4 groups ofresonances from the aromatic region. The resonances at 7.203 (dd, J=8.4Hz, 2.2 Hz) and 6.965 (dd, 8.4 Hz, 2.2 Hz) ppm (peaks B and D in FIG.34) suggested this sample contained 1,4-substituted benzene ring(s). Twomutually coupled single-proton doublet resonances (J=2.2 Hz) at 6.900and 6.970 ppm (peaks E and C in FIG. 34) suggested the existence of1,2,3,5-substituted benzene ring(s). The N-linked protons showed at8.330 ppm (peak A in FIG. 34). Furthermore, the ratio of integrated areaof signals A:B:C:D:E was 12:4:2:4:2. These results strongly suggestedthat the “unknown” sample corresponded to diDT, in which all the N atomswere fully protonated.

By using the ChemDraw program, the predicted data of diDT showed similarspectrum pattern and chemical shifts as those of the “unknown” alsoindicated that it was dilDT (FIG. 35).

Application of the Technology

Example 2 Fiber Formation

Crosslinking extensin modules (SOOOOTOVYK; see Tables 16 and 17) (Note:here onward the single letter codes are used to denote amino acids, withrepresenting Hyp) are placed at the N- and C-termini, and a centralstretch composed of rigid SOOOO repeats (bolded below) flanked on eitherside with elastin sequences (italicized below; See Table 18). Thisconstruction should produce tough, rigid, protease-resistant, elasticfibres of high tensile strength, analogous to fibres found in musselbyssus threads but with enhanced water-holding ability in theglycosylated regions.

Each of the repetitive modules in Examples 2A and 2B, including theelastin module, has already been successfully expressed in plants.Peroxidase-catalyzed intermolecular crosslinking of theThr-Hyp-Val-Tyr-Lys module at the ends of the molecules should producelong fibres. (Note: the Lys residues in P1 extensin sequences areresistant to trypsin).

Example 2A

SOOOOTOVYKSOOOOTOVYKSOOOO[VGVPG VGVPG]₅ [SOOOOSOOOOSOOOO]₆[VGVPGVGVPG]₅- SOOOOTOVYKSOOOOTOVYKSOOOO

The variant below replaces the central rigid, arabinosylated SOOOOrepeats with the more flexible SOSOSO repeats (Bolded below), or AOAOAOrepeats, that serve as arabinogalactan polysaccharide addition sites.These polysaccharide additions sites greatly increase the amount ofcarbohydrate on the molecule, impart a negative charge due to theabundance of uronic acids in the polysaccharide, and promote an extendedconformation.

Example 2B

SOOOOTOVYKSOOOOTOVYKSOOOO[VGVPG VGVPG]₅[SOSOSOSOSOSOSOSOSOSOSOSO]₆[VGVPG VGVPG]₅ SOOOOTOVYKSOOOOTOVYKSOOOO

Example 3 HRGP-Based Emulsifiers

Gum arabic glycoprotein is an excellent emulsifier (J-F Xu and M.Kieliszewski, unpublished data). Features of GAGP as well as other HRGPscan be exploited to create novel emulsifiers. Example 3 is very similarto Example 2, except it lacks crosslinking motifs and the elastinrepeats of Example 2 have been replaced with a smaller (12 residues,italicized below), extended (Pro-rich) but flexible, hydrophobic module.Charge repulsions and steric interference produced by therhamnoglucuronoarabinogalactan polysaccharide sidechains attached to theAOAO repeats should help stabilize an oil in water emulsion bypreventing aggregation/flocculation of the emulsion particles. Furtherstabilization can be achieved by adding crosslinking modules. Variationsinclude glycoproteins having only a single hydrophobic module and asingle glycosylated HRGP module or many of each type dispersed regularlythroughout the chain.

Example 3

VPGVPGVPGVPG[AOAOAOAOAOAOAOAOAOAO]₃ VPGVPGVPGVPG

Example 4 HRGP-Based Emulsifiers with Crosslinking Motifs

This Example exploits variations of the GAGP repeat and the extensinTOVYK intermolecular crosslinking motif (underlined) to create acrosslinked emulsifier. The C-terminus variation of the GAGP repeat(italicized and bolded) has decreased glycosylation, increasedflexibility, and increased hydrophobicity, which will allow the moleculeto interact with the surface of an oil droplet. Such a molecule mightfacilitate slow-release drug delivery from an oil-in-water emulsion.

SOOOTLSOSOTOTOOLGPHSOOOTLSOSOTOTOOLGPHTOVYKSOOOTLSOSOTOTOOLGPHSOOOTLSOSOTOTOOLGPHTOVYKSOOOTLSOSOTOTOOLGPHSOOOTLSOSOTOTOOLGPHTOVYKSOOOTLSOSOTOTOOLGPH

Methods to test the properties of the inventive materials include:

1. Crosslinking—cross-linking of monomers to form multimers can betested several ways:

a. Size exclusion chromatography is an effective assay as alreadydemonstrated by Schnabelrauch et al. (Schnabelrauch, L. S.,Kieliszewski, M. J., Upham, B. L., Alizedeh, H., and Lamport, D. T. A.(1996) Plant J., 9, 477-489.)

b. Assay of free Lys in crosslinks putatively involving Lys residues bythe use of the highly reactive acrylonitrile for the cyanoethylation ofall the freely available —NH₂ groups.

c. Dityrosine or isodityrosine formation for crosslinks involving Tyrresidues (Epstein, L. and Lamport, D. T. A. (1984) Phytochem., 23,1241-1246).

d. Surface hydrophobicity measurements using cis-parinaric acid as afluorescent probe (Liu, M. and Damodaran, S. (1999) J. Agric. FoodChem., 47, 1514-1519; Kato, A. and Nakai, S. (1980) Biochim. Biophys.Acta, 624, 13-20).

2. Mechanical properties of biopolymer films

a. Compression isotherms via Langmuir film balance (Fauconnier, M. L.,Blecker, C., Groyne, J., Razafindralambo, H., Vanzeveren, E., Marlier,M., and Paquot, M. (2000) J. Agric. Food Chem., 48, 2709-2712).

3. Emulsifying properties will be evaluated as follows:

a. At different pH

b. Different ionic strengths (Popineau, Y., Pineau, F., Evon, P., andBerot, S. (1999) Nahrung, 43, 361-367)

c. Stability of the emulsion (Liu, M. and Damodaran, S. (1999) J. Agric.Food Chem., 47, 1514-1519)

d. Droplet size and surface charge: Emulsions containing the smallestglobules tend to be the most stable (200-500 nm), as do emulsions thathave a high surface charge. Droplet size will be measured in a Coultercounter as described by Dickenson et al. (Dickenson, E., Rolfe, S. E.,and Dalgleish, D. G. (1988) Food Hydrocolloids, 2, 397-405). Zetapotential measurements are typically carried out using a Dopplerelectrophoresis apparatus or via moving-boundary electrophoresis(Washington, C. (1990) Int. J. Pharm., 66, 1-21).

4. Fiber formation and Fiber properties—

Extrusion of crosslinked products through an appropriately sized nozzlewill yield fibers. After a curing stage, the physical properties of thefibers will be determined in an extensometer to measure their elasticmodulus and tensile strength (Cosgrove, D. J. (1993) New Phytol., 124,1-23).

5. Elastic properties/viscoelasticity can be measured using a rheometeras described by Larre et al (Larre, C., Denery-Papini, S., Popineau, Y.,Deshayes, G., Desserme, C., and Lefebvre, J. (2000) Cereal Chem. (2000),77, 121-127).

6. Lipid encapsulation—lipid exposed at the surface of a microcapsulecan be measured via extraction with chloroform as in: Minemoto, Y.,Adachi, S., and Matsuno, R. (1999) Food Sci. Technol. Res., 5, 289-293.

TABLE 16 Repetitive Sequences Common in Hydroxyproline-richGlycoproteins Characteristic Sequence HRGPSer-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys P1-type extensinSer-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Hyp-Tyr-Tyr-Tyr-Lys P3extensin Pro-Hyp-Val-Tyr-Lys Proline-rich protein Ala-Hyp-Ala-HypArabinogalactan-protein

TABLE 17 HRGP repetitive glycomodules, peptide modules and theircorresponding properties Repetitive module Properties X-Hyp-Hyp-Hyp-HypGlycomodule common in extensins; this module has a polyproline-IIextended conformation; Extensive arabinosylation of Hyp enhances thepolyproline-II conformation and rigidifies the module (X = Ser, Ala,Thr). X-Hyp-Hyp Glycomodule common in arabinogalactan- proteins; Lesspolyproline II conformation than the X-Hyp-Hyp-Hyp-Hyp glycomodule;First Hyp is always arabinosylated, the second is occasionally. (X =Ser, Ala, Thr) X-Hyp-X-Hyp Glycomodule which defines thearabinogalactan-proteins (i.e. clustered non-contiguous Hyp); Anextended random coil conformation further enhanced byarabinogalactan-polysaccharide addition to each Hyp (X = Ser, Ala).X-Hyp-Val-Tyr-Lys Peptide module of extensins and PRPs; adhesion;peroxidase- catalyzed intermolecular cross-linking; Possible reverseβ-turns; tandem repeats of this module may increase elasticity. (X =Thr, Glu, Pro, His, Ile). Tyr-Tyr-Tyr-Lys Peptide crosslinking modulescommon in extensin; Peroxidase- catalyzed Tyr-Lys-Tyr-Lys (?)intramolecular isodityrosine cross-linking sequences; probableintermolecular cross-link that enhances hydrophobicity; intramolecularcrosslink rigidifies this module. Lys-Pro Ionic/covalent cross-linkingmotif of extensins and PRPs

TABLE 18 Non-HRGP peptide modules and corresponding propertiesThr-Val-Gln-Gln-Glu-Leu Sequences for transglutaminase crosslinkingPro-Gly-Gln-Gln-Ile-Val to Lys residues in HRGPs; formation ofN^(ε)-(γ-glutamyl)lysine. Leu-Cys-Cys-Ser Inter/intramolecular disulfidebond forma- X-Cys-Gly tion. (X = Gln, Lys, Arg). Val-Pro-Gly-Val-GlyElastomeric module

Example 5 Additional Cross-Linking Motifs Summary

P1 extensin is a hydroxyproline-rich glycoprotein (HRGP) that is anintegral part of the primary plant cell wall. It consists mainly of tworepetitive motifs: SPPPPTPVYK and SPPPPVKPYHPTPVYK. Earlier workdemonstrated the requirement of a Val-Tyr-Lys motif for in vitrointermolecular cross-linking of P1 by extensin peroxidase. Tyrosine andlysine contain reactive groups, which are proposed to play a role in thecross-linking of extensin. Here we have tested the role of Val-Tyr-Lysmotif in cross-linking by designing synthetic genes and expressing themin tobacco cells. These genes encode the P1 motif: SOOOOTOVYK (O is Hyp)and variants that differ in their cross-linking motifs withsubstitutions of Tyr→Phe and Lys→Leu. FIG. 36 shows the plasmidconstruct.

The genes were expressed in Nicotiana tabacum BY2 suspension culturedcells as enhanced green fluorescent protein (EGFP) fusion proteins andwere targeted through the ER and Golgi to the cell wall via a tobaccoextensin signal sequence. (FIG. 37 shows photomicrographs of cells.) Thetransgene products were isolated from cell culture medium by acombination of hydrophobic interaction and reverse phase C4 columnchromatography. The fusion proteins underwent hydroxylation of prolineand extensive arabinosylation of Hyp residues similar to endogenous P1.The VYK construct [SPPPPTPVYK]₆ (one of the cell lines expressing thisglycoprotein is designated VYK C1) was cross-linked in vitro by tomatoextensin peroxidase, while the VFL construct [SPPPPTPVFL]₆ (one of thecell lines expressing this glycoprotein is designated VFL-F) showed nocross-linking activity. Another cell line (designated VYL-6.1)expressing 6 repeats of the DNA sequence [SPPPPTPVYL] has not beentested for crosslinking. (FIG. 38 shows results of crosslinkingstudies.)

Experimental Methods

Isolation of the Fusion Proteins

Soluble P1 extensin fusion proteins containing six repeats were isolatedfrom the culture medium. The media was concentrated by rotaryevaporation, dialyzed against water, and then freeze-dried. The drysample was redissolved in 2M sodium chloride and injected onto ahydrophobic interaction column. A step gradient of decreasing sodiumchloride was used to elute the column. Fractions were monitored forfluorescence using a Hewlett-Packard 1100 Series flow-throughfluorometer. The fluorescent fractions were then purified using gelfiltration and reverse-phase chromatography.

In Vitro Cross-Linking Assays of P1 Extensin Gene Products

The isolated fusion proteins were treated with trypsin to remove EGFPand were then tested as substrates for the pl 4.6 peroxidase isolatedfrom tomato. The cross-linking of the monomers was measured by aSuperose 6 gel filtration assay of cross-linked product. Native P1extensin served as the positive control and the (SPPPPTPVFL)₆ served asthe negative control.

Conclusion

Three fusion proteins were expressed in tobacco cell cultures. Theseproteins were isolated from the media using hydrophobic interaction, gelfiltration, and reverse-phase chromatography. Neutral sugar analysis hasshown that the main sugar is arabinose with trace amounts of galactose,which is consistent with native P1 extensin.

The module was cleaved from EGFP by tryptic digestion and the module wasthen tested for cross-linking activity. Native P1 tomato extensin wasused as a control. It appears that the glycoprotein (SOOOOVYK)₆ doescross-link, although to a much lesser extent than P1. The glycoprotein(SOOOOVFL)₆ did not crosslink. The glycoprotein (SOOOOVYL)₆ has not beentested for crosslinking.

Example 6 Cross-Linking HRGPs with Arabinogalactan Glycomodules Overview

The range of crosslinking HRGPs was expanded to include some containingarabinogalactan glycomodules. Thus, we combined theSer-Hyp₄-Ser-Hyp-Ser-Hyp₄-Tyr-Tyr-Tyr-Lys extensin-type arabinosylatedrepeats (abbreviated YK below) that can crosslink with (Ala-Hyp)_(n)arabinogalactan-type repeats (designated AlaPro below). We constructedgenes encoding (AlaPro)₄(YK)₂₀, (AlaPro)₄(YK)₈, and (AlaPro)₄(YK)₄genes, expressed them as EGFP fusion proteins, and tested whether or notthe resulting glycoproteins could be cross-linked by tomato extensinperoxidase.

Methods

1). Design of Oligonucleotides for Constructsa.

Oligonucleotide set for constructing (AlaPro)₄(YK)_(20/8/4)genes-oligo#1

     R   A   P   A   P   A   P   A 5′-C CGG GCT CCA GCA CCT GCC CCAGCC-3′       3′-CGA GGT CGT GGA CGG GGT CGG GGT A-5′    XmaIThe 5′ end of the sense oligonucleotide (encoding the sequence APAPAPA)above contained a XmaI cut site and the 5′ end of the antisenseoligonucleotide contained a BbsI cut site. This allowed insertion of theconstruct into preexisting plasmids pUC-(YK)₂₀, pUC-(YK)₈, andpUC-(YK)₄.

2). Construction of pUC-SS^(tob)(AlaPro)₄(YK)_(20/8/4)EGFP,pUC-SS^(tob)(YK)_(20/8/4)(AlaPro)₃-EGFP andpBl-SS^(tob)(AlaPro)₄(YK)_(20/8/4)EGFP,pBI-SS^(tob)(YK)_(20/8/4)(AlaPro)₄EGFP plasmids.

The oligonucleotide sets were ligated into pUC-(YK)₂₀, pUC-(YK)₈, andpUC-(YK)₄, respectively (See FIG. 39). The resulting(AlaPro)₄(YK)_(20/8/4) and (YK)_(20/8/4)(AlaPro)₃ fragments were ligatedinto pUC-SS^(tob)(AP)₅₁EGFP and replace the (AP)₅₁ gene. TheSS^(tob)(AlaPrO)₄(YK)_(20/8/4)EGFP and SS^(tob)(YK)_(20/8/4)(AlaPro)₃fragments were finally ligated into the plant transformation vectorpBI121 vector to give plasmids: pBI-SS^(tob)(AlaPro)₄(YK)_(20/8/4)EGFPand pBI-SS^(tob)(YK)_(20/8/4)(AlaPro)₄EGFP.

3). Transformation of above gene cassettes into tobacoo BY2 suspensioncultured cells.

Above-modified pBI plasmids were transformed into Agrobacterium;transformants were selected by kanamycin and streptomycin resistance.The agrobacteria were then used to infect suspension cultured tobaccoBY2 cells. The transformed tobacco cells were selected for kanamycinresistance and cultured in SH media for 20 days at room temperature.

4). Fusion protein isolation and purification.

Culture medium from the transformed tobacco cells was collected andconcentrated under vacuum at 28° C. The concentrated media was adjustedwith NaCl to 2M and loaded on a Butyl-sepharose HIC column. The columnwas eluted with a gradient starting with 2M NaCl and ending with water.Green fractions were collected and freeze-dried. The crude fusionproteins were then purified on a Superose-12 preparative gel filtrationcolumn. Green fractions were either repurified on a C-4 reverse phasedsemi-preparative column in a gradient from 0.1% TFA (aq) to 100% of 80%acetonitrile in 0.1% TFA (aq) in 100 minutes, or concentrated anddesalted on centricon spin columns of cutting size of 5 kDa.

5). Removal of EGFP from the isolated fusion proteins.

EGFP was removed from fusion proteins (AlaPro)₄(YK)_(20/8/4)EGFP bytryptic digestion in a solution of 2% ammonium bicarbonate, 5 mM CaCl₂.The glycomodules were isolated from the solution by size exclusionchromatography on the Superose-12 column, and further purified on C-4reverse phased semipreparative column using a 60 min gradient fromstarting with 100% 0.1% TFA (aq) and ending with 80% acetonitrile in0.1% TFA (aq).

6). Hyp-glycoside profiles of fusion proteins.

7.2 mg of (AlaPro)₄(YK)₂₀EGFP was hydrolyzed in 0.44 N NaOH at 105° C.for 18 hr. The hydrolysate was neutralized and freeze-dried.

7). Sugar analysis of fusion proteins.

One-hundred μg of fusion proteins were dissolved in 200 μl of 2N TFA andheated at 121° C. for 2 hrs. The hydrolysate was then reduced andacetylated as described before.

8). Cross-linking reaction of glycomodules and fusion proteins.Cross-linking reactions were carried out in citrate buffer with H₂O₂ andpl 4.6 extensin peroxidase as described before. The reaction mixtureswere then size-fractionated on an analytical Superose-6 column, whichwas monitored for UV absorbance at 220 nm.

Results

1). Sequences of Each Construct

The pUC-derived plasmids were sequenced using a M13 forward primer (seeconstruct sequences in A below).

A. (AP)₄(YK)₂₀EGFP   A   P   A   P   A   P   A   P   S   P   P   P GCTCCA GCA CCT GCC CCA GCC[CCA TCA CCA CCA CCA  P   Y   Y   Y   K   S   P   P   P   P   S   P CCT TAC TAC TAC AAG TCTCCT CCT CCC CCA TCA CCA]₂₀ EGFP B. (AP)₄(YK)₈EGFP  A   P   A   P   A   P   A   P   S   P   P   P GCT CCA GCA CCT GCC CCAGCC[CCA TCA CCA CCA CCA   P   Y   Y   Y   K   S   P   P   P   P   S   PCCT TAC TAC TAC AAG TCT CCT CCT CCC CCA TCA CCA]₈ EGFP C. (AP)₄(YK)₄EGFP  A   P   A   P   A   P   A   P   S   P   P   P GCT CCA GCA CCT GCC CCAGCC[CCA TCA CCA CCA CCA   P   Y   Y   Y   K   S   P   P   P   P   S   PCCT TAC TAC TAC AAG TCT CCT CCT CCC CCA TCA CCA]₄ EGFP

2). Monosaccharide Composition

Neutral sugar analyses and uronic acid assays showed that was mainlyarabinosylated, but also contained Rha, Ara, Gal, and GlcUA.

TABLE 19 Monosaccharide composition of (AP)₄(YK)₂₀EGFP (AP)₄(YK)₂₀EGFPMonosaccharide Molar percentage Rha 1.5 Ara 80.2 Gal 11.7 GlcUA 6.5

3). Hyp-Glycoside Profile.

The Hyp-glycoside profile of (AlaPro)₄(YK)₂₀EGFP showed that theglycoprotein was mainly glycosylated with arabinosides. The molarpercentage of Hyp-polysaccharide was in consistent with the molarpercentage of Hyp flanked by Ala residues to the total Hyp, based on thegene sequence, implying that the Ala-Hyp repeat motifs were the sites ofarabinogalactan polysaccharide addition.

TABLE 20 Hy-glycoside profile of (AlaPro)₄(YK)₂₀EGFP Hyp Type Molar %Hyp-polysaccharide 4.86 Hyp-Ara₄ 45.67 Hyp-Ara₃ 29.49 Hyp-Ara₂ 9.71Hyp-Ara 7.68 Hyp 2.60

4). Cross-Linking.

FIG. 40 shows size exclusion chromatography before (top frame) and after(bottom frame) 30 minutes of crosslinking of (AP)₄(YK)₂₀ catalyzed byextensin peroxidase. Monomeric (AP)₄(YK)₂₀ eluted at 37 minutes in thetop panel. As monomeric (AP)₄(YK)₂₀ was crosslinked, it increased insize. After 30 minutes (AP)₄(YK)₂₀ was polymerized and voided the column(18 min).

FIG. 41 shows chromatographs of the cross-linking reaction of(AP)₄(YK)₂₀EGFP. The upper and lower figures showed the size shift of(AP)₄(YK)₂₀EGFP after crosslinked for 0 minutes and 30 minutes.(AP)₄(YK)₈, and (AP)₄(YK)₄ were also crosslinked by extensin peroxidase,although the rates of crosslinking were slower.

Example 7 Construction of Genes Encoding Cross-Linkable HumanElastin—Arabinogalactan Protein Fusion Proteins Methods

I. Design of Oligonucleotides Encoding the Human Elastin Motifs andRepetitive Ala-Pro.

A set of oligonucleotides was designed based on the codons favored bytobacco cells. The oligonucleotide set encoded four repeats of humanelastin motif Val-Pro-Gly-Val-Gly (Reiersen, et al. 1998) which wereevenly spaced by an AGP motif (three repeats of Ala-Pro) (Tan, et al.2003). The two sticky ends were designed to anneal to the vectorfragments of pUC-(YK)₂ or pUC-(YK)₄ as BbsI and BsmFl fragments (Held,2004). Here, YK stands for the gene encoding the sequence:Ser-Pro-Pro-Pro-Pro-Ser-Pro-Ser-Pro-Pro-Pro-Pro-Tyr-Tyr-Tyr-Lys.

Oligonucleotide Set:

     P   S   G   V   G   V   P   G   V   G     A   P   A   P 5′-CCA TCAGGA GTA GGT[GTT CCA GGA GTT GGC]₂[GCT CCA GCA CCT      3′-GT CCT CATCCA[CAA GGT CCT CAA CCG]₂[CGA GGT CGT GGA Stickyend                   Elastin Motif           AGP motif  A   P   A   G   V   G   V   P   G   V   G GCC CCA GCC]GGT GTT GGA[GTACCT GGT GTT GGT]₂-3′ CGG GGT CGG]CCA CAA CCT[CAT GGA CCA CAA CCA]₂GGTA-5′                               Elastin Motif    Sticky endII. Construction of plasmids pUC-(YK₂-E₂AE₂)₄, pUC-(YK₄-E₂AE₂)₄,pUC-(YK₂-E₂AE₄AE₂)₂, and pUC-(YK₄-E₂AE₄AE₂)₂. (Note: abbreviations E₂AE₂are E: elastin motif; A: AGP motif)

1. Construction of plasmid pUC-E₂AE₂ and pUC-E₂AE₄AE₂

Plasmid pUC-YK₈ was digested by BbsI and BsmFl to remove the YK₈ gene(Held, 2004).

Fifty ng of above set of oligonucleotides were annealed to each other in1× ligase buffer and ligated to the above pUC vector. The formed plasmidwas named pUC-E₂AE₂. The sequence of E₂AE₂ was verified by DNAsequencing.

Plasmid pUC-E₂AE₂ was digested by two sets of restriction enzymesBbsII/ScaI and BsmFl/ScaI, respectively. The 1.1 kb BsmFl-ScaI fragmentand 1.8 kb BbsI-ScaI fragments were ligated to each other to form doublerepeats of the gene cassette E₂AE₂ (FIG. 1) (Held, et al. 2004). Thecorresponding plasmid was named pUC-E₂AE₄AE₂. The sequence of E₂AE₄AE₂was confirmed by DNA sequencing.

FIG. 42 shows a flow chart for gene construction. X represented for thedesired gene. In this project, X can be E₂AE₂, or YK₂, or YK₄. Theligation could be between two same genes, or between two differentgenes.

2. Construction of plasmids pUC-YK₂-E₂AE₂, pUC-YK₄-E₂AE₂,pUC-YK₂-E₂AE₄AE₂, and pUC-YK₄-E₂AE₄AE₂

a). By using the same strategy shown in FIG. 42, the BsmFl-ScaI fragmentof pUC-YK₂ (Held 2004), containing the YK₂ gene was ligated with theBbsI-ScaI fragment of pUC-E₂AE₂ and pUC-E₂AE₄AE₂, respectively, whichled to the formation of pUC-YK₂-E₂AE₂ and pUC-YK₂-E₂AE₄AE₂.

b). As shown in FIG. 42, the BsmFl-ScaI fragment of pUC-YK₄ (Held,2004), containing the YK₄ gene was ligated with the BbsI-ScaI fragmentof pUC-E₂AE₂ and pUC-E₂AE₄AE₂, respectively, which lead to the formationof pUC-YK₄-E₂AE₂ and pUC-YK₄-E₂AE₄AE₂. The purpose of constructing genesencoding different sized cross-linking motifs and elastin/AGP motifs wasto test the differences of physical properties between them. All thegenes were confirmed by DNA sequencing.

3. Construction of plasmids pUC-(YK₂-E₂AE₂)₄, pUC-(YK₄-E₂AE₂)₄,pUC-(YK₂-E₂AE₄AE₂)₂, and pUC-(YK₄-E₂AE₄AE₂)₂.

a). By using the method shown in FIG. 42, pUC-YK₂-E₂AE₂,pUC-YK₂-E₂AE₄AE₂, pUC-YK₄-E₂AE₂, and pUC-YK₄-E₂AE₄AE₂ were dimerized,respectively, with themselves. The desired gene sizes of (YK₂-E₂AE₄AE₂)₂and (YK₄-E₂AE₄AE₂)₂ were about 600 and 800 bps, respectively, which werelong enough for expression by tobacco cells. The corresponding plasmidswere named pUC-(YK₂-E₂AE₄AE₂)₂ and pUC-(YK₄-E₂AE₄AE₂)₂.

b). The obtained pUC-(YK₂-E₂AE₂)₂ and pUC-(YK₄-E₂AE₂)₂ were furtherdimerized by the same method shown above, respectively. The formedpUC-(YK₂-E₂AE₂)₄, pUC-(YK₄-E₂AE₂)₄ had the gene cassettes of sizes about800 and 1200 bps, respectively.

All the sequences were confirmed by DNA sequencing.

III. Construction of plasmids pBI-SS^(tob)-(YK₂-E₂AE₂)₄-EGFP,pBI-SS^(tob)-(YK₄-E₂AE₂)₄-EGFP, pBI-SS^(tob)-(YK₂-E₂AE₄AE₂)₂-EGFP, andpBI-SS^(tob)-(YK₄-E₂AE₄AE₂)₂-EGFP.

1. The constructed gene fragments in pUC-(YK₂-E₂AE₂)₄, pUC-(YK₄-E₂AE₂)₄,pUC-(YK₂-E₂AE₄AE₂)₂, and pUC-(YK₄-E₂AE₄AE₂)₂ were removed from theplasmids by the restriction enzymes XmaI and NcoI, and ligated intopUC-SS^(tob)-(AP)₁₁-EGFP by replacing the (AP)₁₁ fragment (flanked byXmaI and NcoI restriction sites), respectively (Shpak, et al., 1999,2001, Tan, et al 2003, Tan 2003). The four new plasmids were namedpUC-SS^(tob)-(YK₂-E₂AE₂)₄-EGFP, pUC-SS^(tob)-(YK₄-E₂AE₂)₄-EGFP,pUC-SS^(tob)-(YK₂-E₂AE₄AE₂)₂-EGFP, andpUC-SS^(tob)-(YK₄-E₂AE₄AE₂)₂-EGFP, respectively.

2. The gene cassettes in above four new pUC plasmids were furtherremoved from original plasmids by BamHI and SacI, and ligated into pBI121 between the BamHI and SacI restriction sites (Shpak, et al., 1999,2001, Tan, et al 2003, Tan 2003). Then, we obtained the planttransformation plasmids: pBI-SS^(tob)-(YK₂-E₂AE₂)₄-EGFP,pBI-SS^(tob)-(YK₄-E₂AE₂)₄-EGFP, pBI-SS^(tob)-(YK₂-E₂AE₄AE₂)₂-EGFP, andpBI-SS^(tob)-(YK₄-E2AE4AE2)₂-EGFP.

All gene sequences were confirmed by DNA sequencing with a primerderived from tobacco extensin signal sequence.

IV. Transfer the genes into tobacco cells.

1. Each 100 ng of above-modified pBI plasmid was transformed intoAgrobacterium by the Freeze-thaw method (An, et al. 1988). The positivecolonies of agrobacterium were selected by Kanamycin/Streptomycinresistance.

2. Overnight-growed transformed agrobacteria were used to co-culturewith 4-day cultured tobacco cells (in SH media) at 28° C. for 2 days.

3. The infected tobacco cells were washed 4 times with SH media toremove the excess agrobacteria. The tobacco cells were spread on SHsolid plates with Kanamycin (100 μg/ml) and Timentin (200 μg/ml).

The following documents, some of which have been cited and referred toherein, are considered part of this disclosure and are incorporatedherein by reference in their entirety.

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While particular embodiments of the subject invention have beendescribed, it will be obvious to those skilled in the art that variouschanges and modifications of the subject invention can be made withoutdeparting from the spirit and scope of the invention. In addition, whilethe present invention has been described in connection with certainspecific embodiments thereof, it is to be understood that this is by wayof illustration and not by way of limitation and the scope of theinvention is defined by the appended claims which should be construed asbroadly as the prior art will permit.

The disclosure of all patents, patent applications (and any patentswhich issue thereon, as well as any corresponding published foreignpatent applications), and publications mentioned throughout thisdescription are hereby incorporated by reference herein. It is expresslynot admitted, however, that any of the documents incorporated byreference herein teach or disclose the present invention.

It should be understood that every maximum numerical limitation giventhroughout this specification will include every lower numericallimitation, as if such lower numerical limitations were expresslywritten herein. Every minimum numerical limitation given throughout thisspecification will include every higher numerical limitation, as if suchhigher numerical limitations were expressly written herein. Everynumerical range given throughout this specification will include everynarrower numerical range that falls within such broader numerical range,as if such narrower numerical ranges were all expressly written herein.

Except where otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding approaches.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of the ordinaryskill in the art to which this invention belongs. The terminology usedin the description of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention. Asused in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, all of whichare hereby incorporated by reference in their entirety. The embodimentswithin the specification provide an illustration of embodiments of theinvention and should not be construed to limit the scope of theinvention. The skilled artisan recognizes that many other embodimentsare encompassed by the claimed invention and that it is intended thatthe specification and examples be considered as exemplary only, with atrue scope and spirit of the invention being indicated by the followingclaims.

1. A non-naturally occurring protein comprising at least onemodification, wherein the at least one modification comprises theaddition of the amino acid sequence Tyr-X-Tyr, or a substitution ordeletion to result in the presence of Tyr-X-Tyr in the sequence, whereinX is chosen from any amino acid, and wherein the protein, in thepresence of oxidizing conditions, forms at least one of intermolecularcrosslinks and intramolecular crosslinks, and wherein the.
 2. Thenon-naturally occurring protein according to claim 1, wherein X ischosen from Tyr, Lys, and Val.
 3. The non-naturally occurring proteinaccording to claim 1, wherein the amino acid sequence comprisesTyr-X-Tyr-Lys.
 4. The non-naturally occurring protein according to claim1, wherein the protein comprises two or more Tyr-X-Tyr motifs.
 5. Thenon-naturally occurring protein according to claim 1, intermolecularlycrosslinked to a non-naturally occurring protein comprising the aminoacid sequence Tyr-X-Tyr.
 6. The non-naturally occurring proteinaccording to claim 1, intramolecularly crosslinked to itself.
 7. Thenon-naturally occurring protein according to claim 1, further comprisingthe amino acid sequence X-Hyp_(n), wherein X is any amino acid, and n isfrom 1 to about
 1000. 8. The non-naturally occurring protein accordingto claim 7, wherein X is chosen from Ser, Ala, Val, and Thr.
 9. Thenon-naturally occurring protein according to claim 1, further comprisingthe amino acid sequence X-Hyp-X-Hyp, wherein X is any amino acid. 10.The non-naturally occurring protein according to claim 9, wherein X ischosen from Ser, Ala, Val, and Thr.
 11. A non-naturally occurringprotein comprising the amino acid sequence Val-Tyr-Lys.